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CN102906267A - Identification of circadian rhythms in photosynthetic and non-photosynthetic tissues of maize and their use in crop improvement - Google Patents

Identification of circadian rhythms in photosynthetic and non-photosynthetic tissues of maize and their use in crop improvement Download PDF

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CN102906267A
CN102906267A CN2011800121142A CN201180012114A CN102906267A CN 102906267 A CN102906267 A CN 102906267A CN 2011800121142 A CN2011800121142 A CN 2011800121142A CN 201180012114 A CN201180012114 A CN 201180012114A CN 102906267 A CN102906267 A CN 102906267A
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clock
round
corn
plant
polynucleotide
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O.N.丹尼列夫斯卡亚
J.E.黑本
K.R.海斯
C.R.西蒙斯
S.D.德尚
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Pioneer Hi Bred International Inc
EIDP Inc
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EI Du Pont de Nemours and Co
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Abstract

本发明提供涉及玉米的叶和穗组织中昼夜循环的多核苷酸序列。本发明提供了多核苷酸序列及其编码的与振荡相关的多肽的用途。所述公开的序列负责控制作物中植物生长、源-库关系和产量。

Figure 201180012114

The present invention provides polynucleotide sequences involved in the diurnal cycle in leaf and ear tissues of maize. The present invention provides the use of the polynucleotide sequence and its coded polypeptide related to oscillation. The disclosed sequences are responsible for the control of plant growth, source-sink relationships and yield in crop plants.

Figure 201180012114

Description

Photosynthesis of maize tissue and non-photosynthesis are organized circadian evaluation and the purposes aspect Crop Improvement thereof
Technical field
The present invention relates generally to biology field.
Background technology
Diurnal cycle be control the plant daily rhythms and season the rhythm and pace of moving things an essential environmental factors.Light and shade changes the inner circadian clock of drive round the clock, and circadian clock is created in (freely turning round) rhythm and pace of moving things of keeping voluntarily under the constant illumination condition.A simplifying model of circadian clock is comprised of the three basic constituent element: import pathway, its energy sensitization; Core oscillator, it is the mechanism of transcribing that produces the rhythm and pace of moving things; And output pathway, it controls various growths and metabolic process, cause the suitable physiologic adaptation (Barak to the daily cycle, et a1., (2000) Trends Plant Sci 5:517-522 (people such as Barak,, " plant science trend " in 2000, the 5th volume, the 517-522 page or leaf); Harmer, (2009) Annu Rev Plant Biol 60:357-377 (Harmer,, " plant biological academic year comments ", the 60th volume, 357-377 page or leaf in 2009)).Internal clocking and outside bright/dark circulation make synchronously suitably that plant is strongr, survival rate is higher, competitive edge more obvious (Dodd, et al., (2005) Science 309:630-633 (people such as Dodd, 2005, " science ", the 309th volume, 630-633 page or leaf)) and the better (Ni of growing way, et al., (2009) Nature 457:327-331 (people such as Ni,, " nature " in 2009, the 457th volume, the 327-331 page or leaf)).
Up to the present, the gene structure of plant diel rhythm system sets forth at most (Mas, (2008) Trends Cell Biol 18:273-281 (Mas in Arabidopis thaliana, 2008, " cytobiology trend ", the 18th volume, 273-281 page or leaf)).Import pathway is comprised of two cover Photoreceptorss, namely experience the phytochrome (PHYA-E) of ruddiness/far-red light and experience the cryptochrome (CRY1 and CRY2) of UV-A/ blue light, they are in perceived light in the daytime and to core oscillator transmitted signal (Nemhauser, (2008) Curr Opin Plant Biol 11:4-8 (Nemhauser, 2008, " plant biology is newly seen ", the 11st volume, 4-8 page or leaf)).The core oscillator gene forms the chain feedback control loop (Harmer and McClung, (2009) Science 323:1440-1441 (Harmer and McClung,, " science ", the 323rd volume, 1440-1441 page or leaf in 2009)) of transcribing.The daystart loop is comprised of MYB sample transcription factor CCA1 (CIRCADIAN CLOCK ASSOCIATED) and LHY (LATE ELONGATED HYPOCOTYL), and they participate in the regulation and control of two different loops.In the daystart loop, CCA1/LHY negative regulation puppet is replied transcribing of regulatory factor TOC1 (TIMING OF CAB EXPRESSION 1) and TCP sample transcription factor CHE (CCA1 HIKING EXPEDITION).TOC1/CHE forms the mixture (Pruneda-Paz, et al., (2009) Science 323:1481-1485 (people such as Pruneda-Paz,, " science ", the 323rd volume, 1481-1485 page or leaf in 2009)) of just regulating and control CCA1/LHY and transcribing.In loop in the daytime, CCA1/LHY is just regulating and control transcribing of PRR7 and PRR9 (PSEUDO-RESPONSE REGULATORS), and the two is negative regulation CCA1/LHY all.In the night loop, TOC1/CHE serves as the negative regulatory factor of GI (GIGANTIA), and the latter itself is the positive regulatory factor of TOC1.Night gene ZTL (ZEITLUPE, the F-box albumen of protein degradation) degraded of participation TOC1 and PRR3 albumen, be provided on the protein level regulation and control (Mas to the core clock component, et al., (2003) Nature 426:567-570 (people such as Mas,, " nature " in 2003, the 426th volume, the 567-570 page or leaf)).A plurality of chain loops of transcribing had not only kept firmly genetic mechanism but also are no lack of handiness (Harmer (2009)).
Circadian clock produces rhythm and pace of moving things output, thereby regulates and control many development of plants and physiological process, comprising: growth (Nozue, et al., (2007) Nature 448:358-361 (people such as Nozue, 2007, " nature ", the 448th volume, 358-361 page or leaf); Nozue and Maloof, (2006) Plant Cell Environ 29:396-408 (Nozue and Maloof, 2006, " plant, cell and environment ", the 29th volume, the 396-408 page or leaf)), flowering time, the stem tuber of yearly plant forms, the growth of perennial plant stops to form (Lagercrantz with terminal bud, (2009) J Exp Bot 60:2501-2515 (Lagercrantz, 2009, " experimental botany magazine ", the 60th volume, the 2501-2515 page or leaf)), photosynthesis (Sun, et al., (2003) the Plant Mol Biol 53:467-478 (people such as Sun, 2003, " molecular biology of plants ", the 53rd volume, the 467-478 page or leaf)), nitrogen absorbs (Gutierrez, et al., (2008) the Proc Natl Acad Sci USA 105:4939-4944 (people such as Gutierrez, 2008, " institute of NAS periodical ", the 105th volume, the 4939-4944 page or leaf)) and hormone signal conduction and stress reaction (Covington and Harmer, (2007) PLoS Biol 5:e222 (Covington and Harmer, 2007, " Public science Library biology ", the 5th volume, e222 page or leaf).Yet, at present just tentatively understand minute child node (molecular nodes) that circadian clock and output pathway are associated.Up to now, understanding the most clearly, relating dot is the Photoperiod of Arabidopis thaliana and Rice Flowering time.Arabidopis thaliana clock gene GI and paddy rice homologous gene OsGI thereof promote transcription factor CO (CONSTANS) and OsCO (Hd1, HEADING1) expression, bloom (the Michaels that transcribes of incitant FT (FLOWERING LOCUS T) and paddy rice homologous gene Hd3a (HEADING 3a) thereof of the downstream of these two transcription factors control Arabidopis thalianas, (2009) Curr Opin Plant Biol 12:75-80 (Michaels, 2009, " plant biology is newly seen ", the 12nd volume, the 75-80 page or leaf), Tsuji and Komiya, (2008) Rice 1:25-35 (Tsuji and Komiya, 2008, " paddy rice ", the 1st volume, 25-35 page or leaf)).The photoperiod sensitivity approach has been guaranteed to bloom under favourable condition.
There are several pieces of papers to identify that the Arabidopis thaliana core oscillator is related with the molecule between the various plants physiological process.The rhythmicity hypocotyl growth is to be promoted by the positive interaction of two alkaline helix-loop-helix transcription factor PIF4 and PIF5 (PHYTOCHROM-INERACTING FACTOR), their transcriptional level is by CCA1 regulation and control (Nozue, et al., (2007) the Nature 448:358-361 (people such as Nozue, 2007, " nature ", the 448th volume, 358-361 page or leaf)).Hypocotyl growth is also independently regulated and control by the free level of plant hormone growth hormone, growth hormone is produced by growth hormone biosynthesis gene YUCCA8, this gene is directly controlled (Rawat by clock dependency Myb sample transcription factor RVE1 (REVEILLE 1), et al., (2009) Proc Natl Acad Sci USA 106:16883-16888 (people such as Rawat,, " institute of NAS periodical " in 2009, the 106th volume, the 16883-16888 page or leaf)).This is the direct correlation between circadian oscillator and the growth hormone network of regulating and control the Arabidopsis thaliana Seedlings growth.The output pathway of PPR9/7/5 gene and intermediary metabolism (mainly being in the plastosome), particularly tricarboxylic acid (TCA) circulation keeps relevant (Fukushima, et al., (2009) the Proc Natl Acad Sci USA106:7251-7256 (people such as Fukushima, 2009, " institute of NAS periodical ", the 106th volume, 7251-7256 page or leaf)).TOC1 is also with to coerce relevant ABA hormone relevant, it connects (Legnaioli with circadian clock and plant to the reaction of arid, et al., (2009) the The EMBO Journal 28:3745-3757 (people such as Legnaioli, 2009, " European Molecular Bioglogy Organization's proceedings ", the 28th volume, 3745-3757 page or leaf)).
The application of chip technology has disclosed in the Arabidopis thaliana diel rhythm to the generally impact of genetic transcription.These researchs mainly concentrate on sensitization tissue, for example Arabidopis thaliana lotus throne.In chlorenchyma, nearly 35% arabidopsis gene is subjected to diel rhythm regulation and control (Covington, et al., (2008) Genome Biol9:R130 (people such as Covington,, " genome biology ", the 9th volume, R130 page or leaf in 2008); Harmer, et al., (2000) Science 290:2110-2113 (people such as Harmer,, " science ", the 290th volume, 2110-2113 page or leaf in 2000); Ptitsyn, (2008) BMC Bioinformatics 9 (9): S18 (Ptitsyn,, " BMC information biology ", the 9th phase of the 9th volume, S18 page or leaf in 2008)).Although animal model shows that almost the transducer of each tissue has very large diel rhythm component, but not yet to the various plants tissue system assess the round the clock Relative Contribution (Ptitsyn of photoperiod to transcribing, et al., (2006) the PLoS Comput Biol 2:e16 (people such as Ptitsyn, 2006, " Public science Library calculation biology ", the 2nd volume, e16 page or leaf)).In the front genome epoch, in leaf of Semen Maydis photosynthesis and elongate leaf speed, observe round the clock and change, they reach peak value (Kalt-Torres and Huber at noon, (1987) Plant Physiol 83:294-298 (Kalt-Torres and Huber,, " plant physiology " in 1987, the 83rd volume, the 294-298 page or leaf), Kalt-Torres, et al., (1987) the Plant Physiol 83:283-288 (people such as Kalt-Torres, 1987, " plant physiology ", the 83rd volume, the 283-288 page or leaf), Usuda, et al., (1987) Plant Physiol83:289-293 (people such as Usuda, 1987, " plant physiology ", the 83rd volume, 289-293 page or leaf)).In non-photosynthetic seed, also found the round the clock variation of endosperm-specific transcription factor O2 (Opaque 2), and someone proposes the O2 activity by round the clock metabolite flow control (Ciceri, et al., (1999) the Plant Physiol 121:1321-1328 (people such as Ciceri, 1999, " plant physiology ", the 121st volume, 1321-1328 page or leaf)).The corn homologous gene of GI (gigz1) and CO (conz1) has been proved to be diel rhythm, they are direct output (Miller of the circadian clock in the Photoperiod pathway of control Arabidopis thaliana flowering time, et al., (2008) the Planta 227:1377-1388 (people such as Miller, 2008, " plant ", the 227th volume, the 1377-1388 page or leaf)), although Temperate maize is to bloom not to be subjected to the day-neutral plant of length-adjusting on daytime.
Two TOC1 homologous gene ZmTOCa and ZmTOCb have been identified in this research, and they are positioned respectively No. 5 and No. 4 karyomit(e)s.Transcribing all of two genes reaches peak value at 6pm, and this conforms to Arabidopis thaliana TOC1 genetic expression.TOC1 is the member that puppet is replied regulatory factor (PRR) family, this family is by upper five the conservative PRR genomic constitution (Murakami that evolve in Arabidopis thaliana and the paddy rice, et al., (2007) the Biosci Biotechnol Biochem 71:1107-1110 (people such as Murakami, 2007, " bio-science, biotechnology and biological chemistry ", the 71st volume, 1107-1110 page or leaf); Murakami, et al., (2003) Plant Cell Physiol 44:1229-1236 (the village upper class,, " plant cell physiology ", the 44th volume, 1229-1236 page or leaf in 2003)).Except two ZmTOC1 homologous genes, ZmPRR73, ZmPRR37 and ZmPRR59 have also been identified in this research, and they are based on the sequence similarity level according to (the village upper class, (2003)) of paddy rice PRR unnamed gene.Two ZEITLUPE homologous genes (Kim, et al., (2007) Nature 449:356-360 (people such as Kim have also been identified, 2007, " nature ", the 449th volume, the 356-360 page or leaf)) ZmZTLa and ZmZTLb, they are positioned No. 2 and No. 4 karyomit(e)s.Two corn ortholog gene gigz1A of GIGANTIA and gigz1B be the existing (Miller that describes before this, et al., (2008) the Planta 227:1377-1388 (people such as Miller, 2008, " plant ", the 227th volume, the 1377-1388 page or leaf)), confirmed their vibrations in fringe and leaf at this.Agilent (Anjelen Sci. ﹠ Tech. Inc's life science and chemical analysis division department, No. 2850, cent Weir road, Delaware, USA Wilmington city, 19808-1610 (Agilent Technologies, Inc., Life Sciences and Chemical Analysis, 2850 Centerville Road, Wilmington, DE 19808-1610, and hundred million sensible (hundred million sensible limited-liability company USA)), No. 9885, main road, center, town, the inferior San Diego of U.S. markon welfare, 92121 (Illumina, Inc., 9885Towne Centre Drive, San Diego, CA 92121 USA)) analyze and illustrate that all the known core constituent element of major part moves in circles.Analyze the circulation of further having confirmed core component ZmCCA, ZmLHY, ZmTOC1a and ZmTOC1b by RT-PCR.Compare with leaf texture, the amplitude of the core constituent element in the growth period fringe weakens, but still strong.These data show that majority of plant core oscillator system for example organizes at non-photosynthesis and work in the fringe, but vibrator output obviously separates far away with the transcriptional machinery that affect downstream Circadian Expression change.
Core clock mechanism and come from this component of adjacent signal mechanism for example for example leaf and fringe Relations Among reach forward and affect the such mode of crop performance and improve change or extended source and storehouse.Round the clock pattern and the apparent adaptability (Ni, (2009)) of combination have been confirmed to strengthen from the extensive genetic complementation of the round the clock pattern in different germplasms source.
Description of drawings
Fig. 1: round the clock core clock constituent element, the chromosome position that in corn, works and the time that reaches the peak value expression level.
Fig. 2: the Circadian Expression of verifying ZmCCA1, ZmLHY, ZmTOC1a and ZmTOC1b with qRT-PCR.
Fig. 3: the time of the gene of Circadian Expression, chromosome position and peaking expression level in the fringe.
The exon/intron structure of Fig. 4: ZmCCA1 and ZmLHY gene
Fig. 5: according to the round the clock pattern of the gene function item of time enrichment
Summary of the invention
At present also not to corn round the clock/the physiological rhythm transcriptional profile carried out systematic study.Beginning this research work, is in order to transcribe the scope of middle effect at regulatory gene with diurnal cycle in the modern full Genome Atlas analytical technology investigation corn.Design the field experiment under natural undisturbed condition, and gathered the sample of photosynthesis tissue (being leaf) and non-photosynthesis tissue (being the growth period fringe).The thousands of transcripts that in leaf of Semen Maydis, circulate have significantly been identified.Yet in non-photosynthetic fringe, only have few small part gene to 45 to carry out significantly diurnal cycle.Wherein many is corn homologous genes of Arabidopis thaliana core oscillator gene, shows that core diel rhythm gene guards in corn, and in photosynthesis tissue and non-photosynthesis tissue Circadian Expression.
In analysis, identified the gene that a plurality of corns are regulated and control round the clock.471 sequences altogether, comprise from the sequence of prematurity fringe, in leaf texture, have high amplitude/significantly circulation sequence and with NUE and carbon:: the multiple sequence that the nitrogen function is relevant.These sequences comprise polypeptide and the relative promotor of ORF, coding.
Below tabulation comprises some embodiments of the present invention:
1. isolated polynucleotide, the polynucleotide of described separation are selected from:
A. polynucleotide, as determining under default parameters with the GAP algorithm, described polynucleotide be selected from SEQ ID NO:1,2,3,4,5,6,7,8,20,184,186,188,190,192,194,196,198,200,202,204,206,208,210,212,214,216,218,220,222,224,226,228,230,232,234,236,238,240,242,244,246,248,250,252,254,256,258,260,262,264,266,268,270,272,274,276,278,280,282,284,286,288,290,292,294,296,298,300,302,304,306,308,310,312,314,316,318,320,322,324,326,328,330,332,334,336,338,340,342,344,346,348,350,352,354,356,358,360,362,364,366,368,370,372,374,376,378,380,382,384,386,388,390,392,394,396,398,400,402,404,406,408,410,412,414,416,418,420,422,424,426,428,430,432,434,436,438,440,442,444,446,448,450,452,, 454,456,458,460,462,464,466, the full length sequence of 468 and 470 polynucleotide has at least 90% sequence identity; Wherein said polynucleotide encoding polypeptide, described polypeptide has the function of diel movement conditioning agent;
B. polynucleotide, described polynucleotide are selected from SEQ ID NO:1,2,3,4,5,6,7,8,20,184,186,188,190,192,194,196,198,200,202,204,206,208,210,212,214,216,218,220,222,224,226,228,230,232,234,236,238,240,242,244,246,248,250,252,254,256,258,260,262,264,266,268,270,272,274,276,278,280,282,284,286,288,290,292,294,296,298,300,302,304,306,308,310,312,314,316,318,320,322,324,326,328,330,332,334,336,338,340,342,344,346,348,350,352,354,356,358,360,362,364,366,368,370,372,374,376,378,380,382,384,386,388,390,392,394,396,398,400,402,404,406,408,410,412,414,416,418,420,422,424,426,428,430,432,434,436,438,440,442,444,446,448,450,452,, 454,456,458,460,462,464,466, the full length sequence of 468 and 470 polynucleotide has at least 90% sequence identity;
C. with the polynucleotide of (a) or polynucleotide complete complementary (b);
D. by the polypeptide of (a) or polynucleotide encoding (b); With
E. polypeptide, as determining under default parameters with the GAP algorithm, described polypeptide be selected from SEQ ID NO:185,187,189,191,193,195,197,199,201,203,205,207,209,211,213,215,217,219,221,223,225,227,229,231,233,235,237,239,241,243,245,247,249,251,253,255,257,259,261,263,265,267,269,271,273,275,277,279,281,283,285,287,289,291,293,295,297,299,301,303,305,307,309,311,313,315,317,319,321,323,325,327,329,331,333,335,357,359,361,363,365,367,369,371,373,375,377,379,381,383,385,387,389,391,393,395,397,399,401,403,405,407,409,411,413,415,417,419,421,423,425,427,429,431,433,435,437,439,441,443,445,447,449,451,453,455,457,459,461,463,465,467,467, the full length sequence of 469 and 471 polypeptide has at least 90% sequence identity.
2. recombinant expression cassettes, described recombinant expression cassettes comprises polynucleotide claimed in claim 1, and wherein said polynucleotide effectively are connected to promotor with the sense or antisense direction.
3. host cell, described host cell comprises expression cassette claimed in claim 2.
4. transgenic plant, described transgenic plant comprise recombinant expression cassettes claimed in claim 2.
5. transgenic plant according to claim 4, wherein said plant is monocotyledons.
6. transgenic plant according to claim 4, wherein said plant is dicotyledons.
7. transgenic plant according to claim 4, wherein said plant is selected from: corn, soybean, Sunflower Receptacle, Chinese sorghum, canola oil dish, wheat, clover, cotton, paddy rice, barley, millet, peanut, sugarcane and cocoa.
8. transgenic seed, described transgenic seed is from transgenic plant claimed in claim 4.
9. adjust the circadian method of plant for one kind, described method comprises:
A. introduce recombinant expression cassettes in vegetable cell, described recombinant expression cassettes comprises the polynucleotide claimed in claim 1 that effectively are connected to promotor; With
B. under the plant cell growth condition, cultivate described plant; Diel rhythm in the wherein said vegetable cell is modulated.
10. method according to claim 9, wherein said vegetable cell is from being selected from following plant: corn, soybean, Sunflower Receptacle, Chinese sorghum, canola oil dish, wheat, clover, cotton, paddy rice, barley, millet, peanut, sugarcane and cocoa.
11. whole strain plant or circadian method in the modulation plant, described method comprises:
A. introduce recombinant expression cassettes in vegetable cell, described recombinant expression cassettes comprises the polynucleotide claimed in claim 1 that effectively are connected to promotor;
B. under the plant cell growth condition, cultivate described vegetable cell; With
C. from described vegetable cell aftergrowth; Diel rhythm in the wherein said plant is modulated.
12. method according to claim 11, wherein said plant is selected from: corn, soybean, Chinese sorghum, canola oil dish, wheat, clover, cotton, paddy rice, barley, millet, peanut and cocoa.
13. a product that derives from the method that the transgenic plant tissue is processed, the separation polynucleotide of the gene that described transgenic plant tissue expression coding works round the clock, described method comprises:
A. use the recombinant expression cassettes transformed plant cells, described recombinant expression cassettes comprises polynucleotide, described polynucleotide be selected from SEQ ID NO:1,2,3,4,5,6,7,8,20,40,184,186,188,190,192,194,196,198,200,202,204,206,208,210,212,214,216,218,220,222,224,226,228,230,232,234,236,238,240,242,244,246,248,250,252,254,256,258,260,262,264,266,268,270,272,274,276,278,280,282,284,286,288,290,292,294,296,298,300,302,304,306,308,310,312,314,316,318,320,322,324,326,328,330,332,334,336,338,340,342,344,346,348,350,352,354,356,358,360,362,364,366,368,370,372,374,376,378,380,382,384,386,388,390,392,394,396,398,400,402,404,406,408,410,412,414,416,418,420,422,424,426,428,430,432,434,436,438,440,442,444,446,448,450,452,, 454,456,458,460,462,464,466, the full length sequence of 468 and 470 polynucleotide has at least 90% sequence identity; Described polynucleotide are connected to promotor effectively; With
B. under the plant cell growth condition, cultivate the vegetable cell of described conversion; The growth of the vegetable cell of wherein said conversion is modulated;
C. under the condition that forms plant, cultivate described vegetable cell, in plant tissue, to express described polynucleotide; With
D. described plant tissue is processed to obtain product.
14. transgenic plant according to claim 13, wherein said plant is monocotyledons.
15. transgenic plant according to claim 13, wherein said plant is selected from: corn, soybean, Sunflower Receptacle, Chinese sorghum, canola oil dish, wheat, clover, cotton, paddy rice, barley, sugarcane and millet.
16. transgenic plant according to claim 4, the plant-growth that causes comparing with unconverted plant improvement is expressed in crossing of wherein said polynucleotide.
17. transgenic plant according to claim 4, wherein said plant is compared with unconverted plant, shows the Resource of improvement.
18. transgenic plant according to claim 4, wherein said plant is compared with unconverted plant, has the output of raising.
19. comprising, a regulation and control polynucleotide molecule, described molecule be selected from following sequence: (a) SEQ ID NO:31-183; (b) nucleic acid fragment, described nucleic acid fragment comprise at least 50-100 continuous nucleotide of one of SEQ ID NO:31-183, and wherein said fragment comprises the listed one or more round the clock controlling elements of table 2; (c) nucleotide sequence, described nucleotide sequence have the identity with GAP algorithm that determine and about 500-1000 continuous nucleotide at least 90% one of SEQ ID NO:31-183 under default parameters.
20. a chimeric polynucleotide molecule, described molecule comprise the described nucleic acid fragment of claim 19.
21. chimeric molecule according to claim 20, described molecule comprise described round the clock controlling element and tissue specific expression element.
22. chimeric molecule according to claim 21, wherein said tissue specific expression element is selected from root-specific, vascular bundle sheath cell specificity, leaf specificity and embryo-specific expression element.
23. regulation and control polynucleotide molecule according to claim 19, wherein said regulation and control polynucleotide molecule is promotor.
24. a construct that comprises the described regulatory molecule of claim 19, described regulatory molecule effectively is connected to heterologous polynucleotide molecules, and wherein said heterologous molecule is given the proterties of paying close attention to.
25. construct according to claim 24, the wherein said proterties of paying close attention to is selected from drought tolerance, frost resistance, winter hardiness, disease resistance and insect-resistant.
26. construct according to claim 24, wherein said heterologous molecule works in source-Ku metabolism.
27. transgenic plant that transform with the described regulatory molecule of claim 19.
28. transgenic plant according to claim 27, it is monocotyledons.
29. transgenic plant according to claim 27, it is selected from: corn, soybean, canola oil dish, cotton, Sunflower Receptacle, clover, beet, wheat, naked barley, paddy rice, sugarcane, oat, barley, turfgrass, Chinese sorghum, millet, tomato, pigeonpea, vegetables, fruit tree and forage grass.
30. a method that increases plant biomass, described method are included under the control of the described regulatory molecule of claim 19 and express the heterologous polynucleotide of paying close attention to.
31. method according to claim 30, wherein said heterologous polynucleotide are the plant genes of regulating and control round the clock.
32. a method that improves plant abiotic stress patience, described method are included in and express one or more under the control of the described regulatory molecule of claim 19 give the polynucleotide of abiotic stress patience in plant.
33. method according to claim 32, wherein said abiotic stress patience is selected from drought tolerance, frost resistance and winter hardiness.
34. method according to claim 33, the wherein said polynucleotide of giving drought tolerance are expressed under controlling element control, and its peak value is expressed and betided about noon or dusk.
35. method according to claim 33, the wherein said polynucleotide of giving frost resistance or winter hardiness are expressed under controlling element control, and its peak value is expressed and betided about dawn or morning centre.
36. the method that the output that reduces transgene expression suppresses, described method comprise that expression effectively is connected to the transgenosis of regulation and control polynucleotide molecule, described regulation and control polynucleotide molecule comprises and is selected from following sequence: (a) SEQ ID NO:31-183; (b) nucleic acid fragment, described nucleic acid fragment comprise at least 50-100 continuous nucleotide of one of SEQ ID NO:31-183, and wherein said fragment comprises the listed one or more round the clock controlling elements of table 2; (c) nucleotide sequence, described nucleotide sequence have the identity with GAP algorithm that determine and about 500-1000 continuous nucleotide at least 90% one of SEQ ID NO:31-183 under default parameters.
37. a screening relates to the method for the candidate gene of abiotic stress patience, described method comprises (a) evaluation described candidate gene of expression under the one or more candidate genes that show the output inhibition under constructive expression or the tissue specific expression and the regulatory molecule that (b) is guiding the Circadian Expression pattern are controlled.
38. comprising, described method according to claim 37, wherein said regulatory molecule be selected from following sequence: (a) SEQ ID NO:31-183; (b) nucleic acid fragment, described nucleic acid fragment comprise at least 50-100 continuous nucleotide of one of SEQ ID NO:31-183, and wherein said fragment comprises the listed one or more round the clock controlling elements of table 2; (c) nucleotide sequence, described nucleotide sequence have the identity with GAP algorithm that determine and about 500-1000 continuous nucleotide at least 90% one of SEQ ID NO:31-183 under default parameters.
Embodiment
Unless otherwise defined, otherwise all technology used herein and scientific terminology all have the common identical meanings of understanding of those skilled in the art.Unless propose in addition, otherwise this paper adopts or the technology considered is standard method well known to those of ordinary skill in the art.Material, method and example only are exemplary rather than restrictive.Following content provides in illustrational mode, but not is intended to limit the scope of the invention.
Hereinafter more completely describe with reference to the accompanying drawings the present invention, wherein show more of the present invention but be not whole embodiment.In fact, these disclosures can multiple multi-form presenting, and should not be construed as and be subject to embodiment as herein described; On the contrary, the purpose that provides of these embodiment is to make the present invention satisfy applicable legal requirements.Similarly numbering refers to similar key element in the text.
By the instruction that provides in the description of front and the accompanying drawing of enclosing, these disclosures those skilled in the art will expect many modification and other embodiment of disclosure described herein.Therefore, should understand, these disclosures are not limited to disclosed specific embodiment, and are intended to modification and other embodiment are included in the scope of this paper end claims.Although this paper has adopted particular term, they only use with general and descriptive sense and not for limiting purpose.
Except as otherwise noted, otherwise enforcement of the present invention will be adopted the routine techniques of phytology, microbiology, tissue culture, molecular biology, chemistry, biological chemistry and recombinant DNA technology, and these technology are in the skill of this area.This class technology has complete explanation in the literature.Referring to for example Langenheim and Thimann, BOTANY:PLANT BIOLOGY AND ITS RELATION TO HUMAN AFFAIRS, John Wiley (1982) (Langenheim and Thimann, " phytology: plant biology and with the relation of human affairs ", John Wei Li press, nineteen eighty-two); CELL CULTURE AND SOMATIC CELL GENETICS OF PLANTS, vol.1, Vasil, ed. (1984) (Vasil edits, 1984 for " culture plant cell and somatic cell genetics ", the 1st volume); Stanier, et al., THE MICROBIAL WORLD, 5 ThEd., Prentice-Hall (1986) (people such as Stanier, " microbial world 1 ", the 5th edition, Prentice Hall press, 1986 years); Dhringra and Sinclair, BASIC PLANT PATHOLOGY METHODS, CRC Press (1985) (Dhringra and Sinclair, " plant pathology basic skills ", CRC press, 1985); Maniatis, et al., MOLECULAR CLONING:A LABORATORY MANUAL (1982) (people such as Maniatis, " molecular cloning experiment guide ", nineteen eighty-two); DNA CLONING, vols.I and II, Glover, ed. (1985) (Glover edits, 1985 for " dna clone ", I volume and II volume); OLIGONUCLEOTIDE SYNTHESIS, Gait, ed. (1984) (" oligonucleotide is synthetic ", Gait edits, 1984); NUCLEIC ACID HYBRIDIZATION, Hames and Higgins, eds. (1984) (" nucleic acid hybridization ", Hames and Higgins edit, 1984) and book series METHODS IN ENZYMOLOGY, Colowick and Kaplan, eds, Academic Press, Inc., San Diego, CA (" Enzymology method ", Colowick and Kaplan edit, academic press, San Diego, California).
The form that unit, prefix and symbol can their SI be accepted represents.Except as otherwise noted, nucleic acid is write from left to right with 5 ' to 3 ' direction; And aminoacid sequence is write to the direction of carboxyl from left to right with amino.Numerical range comprises the numeral that limits this scope.The trigram symbolic representation that amino acid can be known usually by them in this article or the one-letter symbol of recommending by IUPAC-IUB commission on Biochemical nomenclature (IUPAC-IUB Biochemical Nomenclature Commission) represent.Equally, Nucleotide can represent by the one-letter code that they are accepted usually.The term that the below defines is by carrying out more complete definition with reference to this specification sheets integral body.
When description is of the present invention, the term below will adopting, and the definition shown in adopting hereinafter.
So-called " microorganism " means any microorganism (comprising eukaryotic microorganisms and prokaryotic micro-organisms), such as fungi, yeast, bacterium, actinomycetes, algae and protozoon and other unicellular structures.
So-called " amplification " mean to make up nucleotide sequence a plurality of copies or with a plurality of copies of this nucleic acid array complementation, this structure is to utilize in the described nucleotide sequence at least one to carry out as template.Amplification system comprises polymerase chain reaction (PCR) system, ligase chain reaction (LCR) system, based on the amplification (NASBA of nucleotide sequence, Mississauga, the Ontario Canada (Cangene of genome company, Mississauga, Ontario)), Q-β replicative enzyme system, based on the amplification system of transcribing (TAS) and strand displacement amplification (SDA).Referring to for example DIAGNOSTIC MOLECULAR MICROBIOLOGY:PRINCIPLES AND APPLICATIONS, Persing, et al., eds., American Society for Microbiology, Washington, DC (1993) (" diagnosis molecular microbiology: principle and application ", the people such as Persing edit, " AAM ", Washington, 1993).The product of amplification is called amplicon.
Term " conservative modify variant " be applicable to simultaneously aminoacid sequence and nucleotide sequence the two.With regard to specific nucleic acid sequence, the conservative variant of modifying refers to those nucleic acid identical or the conservative variant of modifying of encoding amino acid sequence.Since the degeneracy of genetic code, the identical any given protein of nucleic acid encoding on a large amount of functions.For example, codon GCA, GCC, GCG and GCU this seed amino acid of L-Ala of all encoding.Thereby, in each position by codon regulation L-Ala, can be with this codon alterations described corresponding codon any and can not change coded polypeptide.This nucleic acid variation is " silent variant ", and representative is guarded and modified a kind of of variation.Every kind of nucleotide sequence of this paper of coded polypeptide has also been described every kind of possible silent variant of this nucleic acid.Those of ordinary skill will recognize that (except AUG, it is unique password of methionine(Met) normally for each codon in the nucleic acid; An exception is micrococcus rubens (Micrococcus rubens), Methionine codon (Ishizuka for its GTG, et al., (1993) J.Gen.Microbiol.139:425-32 (people such as Ishizuka, 1993, " general microbiology magazine ", the 139th volume, 425-432 page or leaf)) can be modified to obtain the identical molecule of function.Therefore, the variation of every kind of concealment of the nucleic acid of code book invention polypeptide all lies in every kind of described peptide sequence and incorporates by reference this paper into.
For aminoacid sequence, the technician will recognize, the meeting that nucleic acid, peptide, polypeptide or protein sequence are made changes, adds or lacks single amino acids or amino acid whose each displacement of sub-fraction, disappearance or the interpolation in the coded sequence, when this change causes amino acid to be replaced by amino acid like the chemical classes, be " the conservative variant of modifying ".Thereby, the variable amino-acid residue that is selected from any number of 1 to 15 integer also.Thereby, for example, can make 1,2,3,4,5,7 or 10 change.The conservative variant of modifying provides the not modified similar biological activity of peptide sequence that is derived from them usually.For example, substrate specificity, enzymic activity or ligand/receptor are in conjunction with being generally native protein at least 30%, 40%, 50%, 60%, 70%, 80% or 90% of its natural substrate, preferred 60-90%.It is known in the art that amino acid whose conservative substitution table similar on the function is provided.
Six following groups respectively contain the amino acid for being each other conservative substitution:
1) L-Ala (A), Serine (S), Threonine (T);
2) aspartic acid (D), L-glutamic acid (E);
3) l-asparagine (N), glutamine (Q);
4) arginine (R), Methionin (K);
5) Isoleucine (I), leucine (L), methionine(Met) (M), α-amino-isovaleric acid (V); With
6) phenylalanine (F), tyrosine (Y), tryptophane (W).
In addition referring to Creighton, PROTEINS, W.H.Freeman and Co. (1984) (Creighton, " protein ", W.H. freeman company, 1984).
As used herein, " basically by ... form " mean can comprise extra sequence at herbicide-tolerant polynucleotide in the following situation: this extra sequence can optionally not hybridized the identical cDNA to the cDNA of hybridizing with these polynucleotide under stringent hybridization condition, and this hybridization conditions is included in the washing step that carries out in 0.1X SSC and 0.1% sodium lauryl sulphate under 65 ℃.
With regard to the nucleic acid of appointment, so-called " coding " means to comprise the information of specifying protein of translating into.The nucleic acid of coded protein can comprise non-translated sequence (for example intron) in the translation district of this nucleic acid, maybe can lack this non-translated sequence (for example, as in cDNA) between two parties.The information of coded protein is determined by the son that accesses to your password according to this.Usually, aminoacid sequence is encoded by nucleic acid utilization " general " genetic code.Yet, when using for example certain plants, animal and fungi plastosome, bacterium mycoplasma capri (Mycoplasma capricolum) (Yamao, et al., (1985) the Proc.Natl.Acad.Sci.USA 82:2306-9 (people such as Yamao, 1985, " institute of NAS periodical ", the 82nd volume, the 2306-2309 page or leaf)) or during ciliate macronucleus express nucleic acid, can use the variant of the universal code that exists in these organisms.
When by synthesis method preparation or when changing nucleic acid, can utilize the expection host's of express nucleic acid known codon preference therein.For example, although nucleotide sequence of the present invention all can be expressed in monocotyledons species and dicotyledons species, but can modify to solve to sequence the sub-Preference of specific cryptosystem and the GC content Preference of monocotyledons or dicotyledons, because these Preferences have been proved difference (Murray, et al., (1989) the Nucleic Acids Res.17:477-98 (people such as Murray, " nucleic acids research ", the 17th volume, the 477-498 page or leaf), the document is incorporated herein by reference).Thereby the preferred codon of concrete amino acid whose corn can be drawn by the known sequence from corn.Use about the corn codon from 28 kinds of genes of maize plant and in the table 4 of the people such as Murray (ibid), to list.
As used herein, for " allos " of nucleic acid for originating from the nucleic acid of alien species, perhaps, if originate from same species, then for by premeditated human intervention to its natural form form and/or locus aspect carrying out the substantive nucleic acid of modifying.For example, the promotor that effectively is connected to allos structure gene is the species from the species that are different from this structure gene of deriving, and perhaps if from identical species, then one or both has been carried out substantial modification by its original form.Heterologous protein can originate from alien species, perhaps, if originate from same species, then by premeditated human intervention its natural form has been carried out the essence modification.
So-called " host cell " means to contain carrier and supports the cell that copies and/or express of this expression vector.Host cell can be prokaryotic cell prokaryocyte such as intestinal bacteria, or eukaryotic cell such as yeast, insect, plant, Amphibians or mammalian cell.Preferably, host cell is monocot plant cell or dicotyledons cell, includes but not limited to corn, Chinese sorghum, Sunflower Receptacle, soybean, wheat, clover, paddy rice, cotton, canola oil dish, barley, millet and tomato.Particularly preferred unifacial leaf host cell is the corn host cell.
Term " hybridization complex " comprises the double-strandednucleic acid structure that refers to that the single-chain nucleic acid sequence by two mutual selective cross forms.
Nucleic acid is being inserted in the linguistic context of cell, term " introducing " means " transfection " or " conversion " or " transduction ", comprise and refer to nucleic acid is incorporated in eucaryon or the prokaryotic cell prokaryocyte, wherein this nucleic acid can be incorporated in the genome (for example karyomit(e), plasmid, plastid or Mitochondrial DNA) of cell, change into self-replicating or transient expression (for example, the mRNA of transfection).
Term " separation " refers to the material such as nucleic acid or protein, and this material in fact or be substantially free of usually accompanying or component interactional with it with it of finding in its naturally occurring environment.The material that separates is optional to comprise the material of not finding in its natural surroundings therewith.As defined herein, " separation " nucleic acid is also referred to as " allos " nucleic acid.Unless otherwise prescribed, otherwise term " round the clock nucleic acid " means to comprise the round the clock nucleic acid of the polynucleotide of polypeptide (" round the clock polynucleotide ") of encoding.
As used herein, " nucleic acid " comprises deoxyribonucleotide or the ribonucleoside acid polymer that refers to strand or double chain form, unless and in addition restriction, otherwise contain the known analogue of the essential property that has in the following areas natural nucleotide: it is hybridized to single-chain nucleic acid in the mode similar to naturally occurring Nucleotide (for example peptide nucleic acid(PNA)).
The DNA that so-called " nucleic acid library " means to separate or the set of RNA molecule, the genomic whole part of being transcribed that it comprises and organism is specified in representative basically.The molecular biology book of reference of the standard that is structured in of exemplary nucleic acid library such as genomic library and cDNA library has instruction, such as Berger and Kimmel, GUIDE TO MOLECULAR CLONING TECHNIQUES (Berger and Kimmel, " molecule clone technology guide "), be selected from book series METHODS IN ENZYMOLOGY, vol.152, Academic Press, Inc., San Diego, CA (1987) (" Enzymology method ", the 152nd volume, the academic press, San Diego, California, 1987); Sambrook, et al., MOLECULAR CLONING:A LABORATORY MANUAL, 2 NdEd., vols.1-3 (1989) (people such as Sambrook, " molecular cloning experiment guide ", second edition, the 1-3 volume, 1989) and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, et al., eds, Current Protocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley ﹠amp; Sons, and Inc. (1994 Supplement) (" up-to-date experimental methods of molecular biology compilation ", the people such as Ausubel edit, and are selected from " lab guide ", Green publishes the co-partnership company of affiliated company and John Wei Li father and son publishing company).
As used herein, " effectively connect " comprises the functional connection that refers between First ray (such as promotor) and the second sequence, and wherein promoter sequence is initial or mediate the transcribing of DNA of corresponding the second sequence.In general, it is continuous effectively connecting the nucleotide sequence that means to be connected, and connects if necessary two protein coding regions, is continuous and in identical reading frame.
As used herein, term " plant " comprises the whole plant of finger, plant organ (such as leaf, stem, root etc.), seed and vegetable cell and their filial generation.As used herein, vegetable cell includes but not limited to seed suspension culture, embryo, meristem zone, callus, leaf, root, seedling, gametophyte, sporophyte, pollen and sporule.The floristics that can be used for the inventive method is usually the same wide in range with the higher plant kind that is applicable to transformation technology, comprise monocotyledons and dicotyledons, comprise the kind with the subordinate: Cucurbita (Cucurbita), rose (Rosa), Vitis (Vitis), white walnut (Juglans), Fragaria (Fragaria), Lotus (Lotus), Medicago (Medicago), donkey food grass belongs to (Onobrychis), Trifolium (Trifolium), Trigonella (Trigonella), Vigna (Vigna), both citrus (Citrus), linum (Linum), Geranium (Geranium), cassava (Manihot), Daucus (Daucus), Arabidopsis (Arabidopsis), Btassica (Brassica), Rhaphanus (Raphanus), sinapsis alba belongs to (Sinapis), Atropa (Atropa), Capsicum (Capsicum), Datura (Datura), poison tobacco (Hyoscyamus), tomato belongs to (Lycopersicon), Nicotiana (Nicotiana), Solanum (Solanum), green winter Solanum (Petunia), Digitalis (Digitalis), Ma Zhucao belongs to (Majorana), Cichorium (Ciahorium), Helianthus (Helianthus), Lactuca (Lactuca), Brome (Bromus), Asparagus (Asparagus), antirrhinum (Antirrhinum), hemerocallis (Heterocallis), Nemesis, Pelargonium (Pelargonium), Panicum (Panieum), Pennisetum (Pennisetum), Ranunculus (Ranunculus), Senecio (Senecio), salpiglossis belongs to (Salpiglossis), Cucumis (Cucumis), the magnificent genus in cloth Lip river (Browaalia), Glycine (Glycine), Pisum (Pisum), Phaseolus (Phaseolus), lolium (Lolium), Oryza (Oryza), Avena (Avena), Hordeum (Hordeum), Secale (Secale), allium (Allium) and Triticum (Triticum).Particularly preferred plant is corn.
As used herein, " output " comprises when referring to results the bushel number that has carried out revised every acre of cereal crop for grain moisture (normally 15%).Grain moisture is to measure in the grain in when results.The adjustment of grain test afterwards weight be confirmed as to when results the grain moisture content done every bushel pound weight after revising.As used herein, " source-Ku " of improvement relation comprise refer to grain milk in the supply (being the source) of the assimilate characteristic relevant with the improvement of the ratio between demand (being the storehouse).
As used herein, " polynucleotide " comprise finger ribodesose polynucleotide, ribose polynucleotide or its analogue, described analogue has the essential property of natural nucleus sugar nucleotide aspect following: the nucleotide sequence that its nucleotide sequence of hybridizing and naturally occurring Nucleotide are hybridized under stringent hybridization condition is substantially the same, and/or can translate into the identical amino acid of amino acid of translating with naturally occurring Nucleotide.Polynucleotide can be natural or the full length sequence of allos structure gene or regulatory gene or its subsequence.Except as otherwise noted, otherwise this term comprises sequence and the complementary sequence thereof that refers to appointment.Thereby, for stability or for other reasons be " polynucleotide " that mean at this paper such as this term to DNA or the RNA that main chain has carried out modifying.In addition, comprising the DNA of rare base (such as inosine) or modified base (such as the base of tritylation) (only lifting two examples) or RNA is polynucleotide (such as these terms used herein).Should be appreciated that DNA and RNA carried out a variety of modifications that many useful purposes well known by persons skilled in the art are played in these modifications.Term polynucleotide used herein are contained this class of polynucleotide through the form of chemically modified, enzyme modification or metabolism modification, and the chemical species of virus and the distinctive DNA of cell (comprising particularly simple cell and complex cell) and RNA.
Term " polypeptide ", " peptide " and " protein " are used interchangeably in this article, refer to the polymkeric substance of amino-acid residue.These terms are applicable to wherein one or more amino-acid residues and are the aminoacid polymers of corresponding naturally occurring amino acid whose artificial chemistry analogue, and are applicable to naturally occurring aminoacid polymers.
As used herein, " promotor " comprise refer to DNA in the upstream of transcription initiation and relate to RNA polymerase and the identification of other protein (for example transcription factor) and in conjunction with initial zone of transcribing." plant promoter " is the promotor of transcribing that can cause in vegetable cell.Exemplary plant promoter includes but not limited to from plant, plant virus and is included in those promotors that the bacterium of the gene of expressing the vegetable cell obtains, described bacterium such as Agrobacterium (Agrobacterium) or root nodule bacterium (Rhizobium).Example is preferential initial in some tissue, for example promotor of transcribing in leaf, root, seed, fiber, xylem vessel, test-tube baby or the sclerenchyma.This promotor is called as " the tissue preference "." cell type " specificity promoter mainly drives the expression in some cell type in one or more organs, for example the dimension tube cell in root or the leaf." induction type " or " regulation type " promotor is the promotor that is under the environment control.The example that can realize the envrionment conditions of transcribing of being undertaken by inducible promoter comprises the existence of oxygen free condition or light.The promotor of another type is to grow to regulate promotor, for example drives the promotor of expressing in the pollen development process.Organize preferred, cell type-specific, as the to grow adjusting promotor with induction type to consist of " non-composing type " promotor classification." composing type " promotor is promotor active under most of envrionment conditionss.
As used herein, " controlling element " or " regulation and control polynucleotide " refers to adjust the nucleic acid fragment of the expression of the transcribed polynucleotide relevant with described controlling element.This association may occur with cis.Plant promoter also can be with the controlling element of the expression that adjusts the specific gene that effectively is connected to described promotor.When effectively being connected to transcribed polynucleotide molecule, controlling element affects the transcriptional profile of described transcribed polynucleotide molecule." cis element " or " cis-acting elements " refers to affect the transcriptional regulatory element of the cis acting of genetic expression.Cis element can have in conjunction with adjusting the transcription factor of transcribing, the function of trans-acting protein.One or more round the clock cis elements of gene expression pattern that provide can be provided round the clock promotor disclosed herein.
Plant promoter disclosed herein and controlling element can comprise the nucleotide sequence that produces by the promotor engineering, namely make up the promotor of artificial to produce, synthetic, the chimeric or hybridization of known promotor and/or controlling element.These promotors also can make up the cis element from one or more promotors, for example by add allos tissue specificity controlling element to the promotor that comprises the Circadian Expression controlling element.Therefore, imagine the cis element that comprises at least one promotor disclosed herein, be used for adjusting effectively design, structure and the use of the chimeric or hybrid promoters of the expression of the polynucleotide sequence of connection.
Promoter sequence disclosed herein (comprise SEQ ID NO:31-183 and fragment thereof, 50, for example comprise 100,150,200,300,400,500,600,700,800,900,1000,1100,1200,1300,1400,1500,1600,1700,1800,1900,2000 and maximum 2500 continuous nucleotides and have about 80% or 85% or 90% or 95% or 99% identity with these fragments) expection is used for adjusting the expression pattern of one or more heterologous genes.Term under this linguistic context " allos " refers to that the expression of the Nucleotide paid close attention to is by the promoter sequence or its fragment adjustment that are not described Nucleotide self promotor.According to guidance provided herein, those of ordinary skills can easily prepare the disappearance construct of a plurality of promoter sequences disclosed herein.About 25-50 the continuous nucleotide that selection is positioned at disclosed controlling element 3 ' or 5 ' the distolateral wing is used for adjusting genetic expression.Also carried out mutation analysis to strengthen the round the clock specificity of regulation and control.
Term " round the clock polypeptide " refers to one or more aminoacid sequence.This term also comprises its fragment, variant, homologue, allelotrope or precursor (for example former front albumen or front albumen)." round the clock protein " comprises round the clock polypeptide.Unless otherwise prescribed, otherwise term " round the clock nucleic acid " means to comprise the round the clock nucleic acid of the polynucleotide of polypeptide (" round the clock polynucleotide ") of encoding.
As used herein, " restructuring " comprises cell or the carrier that finger has been modified by introducing heterologous nucleic acids, perhaps comes from the cell through the cell of such modified.Thereby for example, reconstitution cell is expressed not the gene that the intracellular same form with natural (non-restructuring) form exists, or expresses originally unconventionality expression because of premeditated human intervention, expresses natural gene not enough or that do not express.As used herein, term " restructuring " is not contained by natural event (for example spontaneous mutation, natural conversion/transduction/swivel base) cell that carries out or the change of carrier, and described event is those that for example occur in the situation of not deliberating human intervention.
As used herein, " recombinant expression cassettes " is the nucleic acid construct that passes through recombination method or synthesis method generation with a series of regulation nucleic acid elements, and it allows specific nucleic acid at the target cell transcription.Recombinant expression cassettes can be incorporated in plasmid, karyomit(e), Mitochondrial DNA, plastid DNA, virus or the nucleic acid fragment.Usually, except other sequence, the recombinant expression cassettes of expression vector part also comprises nucleic acid to be transcribed and promotor.
Term " residue " or " amino-acid residue " or " amino acid " are used interchangeably in this article, refer to incorporate into the amino acid in protein, polypeptide or the peptide (general designation " protein ").Amino acid can be naturally occurring amino acid, unless limit in addition, can be to bring into play the known analogue of natural amino acid of function with mode like the naturally occurring amino acids otherwise can contain.
Term " selective cross " comprise degree that the degree that refers to nucleotide sequence and the nucleic acid target sequence hybridization of appointment under stringent hybridization condition hybridizes than itself and non-target sequence can detect higher (for example, at least 2 times to background), and basically get rid of non-target nucleic acid.The sequence of selective cross has approximately at least 40% sequence identity usually mutually, the sequence identity of preferred 60-90%, most preferably 100% sequence identity (namely complementary).
Term " stringent condition " or " stringent hybridization condition " comprise the degree that refers to probe and the hybridization of its target sequence will can detect than it and the degree of other sequence hybridizations the condition of higher (for example, at least 2 times to background).Stringent condition is sequence dependent, will be different in different environment.By the severity of control hybridization and/or wash conditions, can differentiate the target sequence (homology detection) that can be up to 100% complementation with probe.Perhaps, can regulate stringency to allow in the sequence some mispairing being arranged, so that detect the similarity (allos detection) than low degree.Preferably, probe is long to be about 500 Nucleotide, but vary in length is very large, from less than 500 Nucleotide to the whole length that equals target sequence.
Usually, stringent condition will be lower than about 1.5M sodium ion for salt concn wherein, be generally about 0.01 to 1.0M Na ion concentration (or other salt), pH is 7.0 to 8.3, to short probe (for example, 10 to 50 Nucleotide) temperature is at least 30 ℃, and long probe (for example surpassing 50 Nucleotide) temperature is at least about those conditions of 60 ℃.Stringent condition also can be realized by adding destabilizing agent such as methane amide or Denhardt ' s.Exemplary low stringency is included in 37 ℃ of lower buffered soln hybridization with 30 to 35% methane amides, 1M NaCl, 1%SDS (sodium lauryl sulphate), and in washing in 1X to 2X SSC (20X SSC=3.0M NaCl/0.3M trisodium citrate) under 50 to 55 ℃.Exemplary medium stringency comprises under 37 ℃ and hybridizing in 40 to 45% methane amides, 1M NaCl, 1%SDS, and washs in 0.5X to 1X SSC under 55 to 60 ℃.Exemplary high stringent condition is included in hybridization under 37 ℃ among 50% methane amide, 1M NaCl, the 1%SDS, and washing under 60-65 ℃ in 0.1X SSC.Washing after specificity is decided by to hybridize usually, key factor are ionic strength and the temperature of final washing soln.For DNA-DNA crossbred, T mCan be by Meinkoth and Wahl, the equation T of (1984) Anal.Biochem.138:267-84 (Meinkoth and Wahl,, " analytical biochemistry ", the 138th volume, 267-284 page or leaf in 1984) m=81.5 ℃+16.6 (%GC)-0.61, (log M)+0.41 (%form)-500/L estimates; Wherein M is the volumetric molar concentration of univalent cation, and %GC is the per-cent of guanylic acid and cytidylic acid(CMP) among the DNA, and %form is the per-cent of methane amide in the hybridization solution, and L is the length (unit is base pair) of crossbred.T mTemperature (under the ionic strength of determining and pH) when being the probe hybridization of 50% complementary target sequence and Perfect Matchings.Per 1% mispairing, T mReduce about 1 ℃; Therefore, can regulate T m, hybridization and/or wash conditions be with the sequence of hybridization to required identity.For example, if seek to have 〉=sequence of 90% identity, then can be with T mReduce by 10 ℃.Usually, stringent condition is chosen as than particular sequence and complementary sequence thereof at the ionic strength of determining and the pyrolysis chain temperature (T under the pH m) low about 5 ℃.Yet extreme stringent condition can adopt specific heat melting temperature(Tm) (T m) low 1,2,3 or 4 ℃ hybridization and/or washing; The appropriateness stringent condition can adopt specific heat melting temperature(Tm) (T m) low 6,7,8,9 or 10 ℃ hybridization and/or washing; Low stringency condition can adopt specific heat melting temperature(Tm) (T m) low 11,12,13,14,15 or 20 ℃ hybridization and/or washing; Utilize this formula, hybridization and washing to form and required T m, those of ordinary skill will recognize that the variation of the severity of hybridization and/or washing soln has obtained description inherently.If required mispairing degree causes T mBe lower than 45 ℃ (aqueous solution) or 32 ℃ (formamide soln), then preferably increase SSC concentration so that can use higher temperature.The detailed guide of related nucleic acid hybridization can find in such as Publication about Document: Tijssen, LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY--HYBRIDIZATION WITH NUCLEIC ACID PROBES, part I, chapter 2, " Overview of principles of hybridization and the strategy of nucleic acid probe assays; " Elsevier, New York (1993) (Tijssen, " biological chemistry and molecular biology experiment technology-with the hybridization of nucleic acid probe ", part i, the 2nd chapter, " about the summary of Hybridization principle and nucleic acid probe analysis strategy ", like to think only that, New York, 1993) and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, chapter 2, Ausubel, et al., eds, Greene Publishing and Wiley-Interscience, New York (1995) (" up-to-date experimental methods of molecular biology compilation ", the 2nd chapter, the people such as Ausubel edit, Green press and Wiley-Interscience, New York, nineteen ninety-five).Unless otherwise prescribed, otherwise in present patent application, high severity is defined as at 4X SSC, 5X Denhardt ' s (5g Ficoll, the 5g polyvinylpyrrolidone, the 5g bovine serum albumin is in 500ml water), in the salmon sperm DNA that boils of 0.1mg/ml and the 25mM sodium phosphate 65 ℃ of lower hybridization, and in 0.1X SSC, 0.1%SDS 65 ℃ of lower washings.
As used herein, " transgenic plant " comprise the plant that refers to comprise heterologous polynucleotide in its genome.In general, heterologous polynucleotide stably is incorporated in the genome so that these polynucleotide are delivered to the successive generation.Heterologous polynucleotide can be integrated into separately in the genome, and perhaps the part as recombinant expression cassettes is integrated in the genome." transgenosis " is used for comprising cell, clone, callus, tissue, plant part or the plant that any its genotype has been changed because of the existence of heterologous nucleic acids in this article, comprises transgenosiss of those initial so changes and those are by carrying out the transgenosis that sexual hybridization or vegetative propagation produce from initial transgenosis.As used herein, term " transgenosis " do not contain by the conventional plant breeding method or by infect such as at random cross fertilization, non-recombinant virus, the change of non-recombinant bacteria transforms, spontaneous generation event non-restructuring swivel base or the spontaneous mutation causes genome (chromogene group or karyomit(e) alia gene group).
As used herein, " carrier " comprises the nucleic acid that refers to be used for transfection host cell and can insert therein polynucleotide.Carrier usually is replicon.Expression vector allows to insert transcribed nucleic acid wherein.
Following term is used for the sequence relation between two or more nucleic acid of explanation or polynucleotide or the polypeptide: (a) " reference sequences ", (b) " comparison window ", (c) " sequence identity ", (d) " sequence identity percentage ratio " and (e) " basically complete same ".
As used herein, " reference sequences " is the sequence of determining as the sequence benchmark.Reference sequences can be the subset of specified sequence or all; The for example fragment of full-length cDNA or gene order or complete cDNA or gene order.
As used herein, " comparison window " means to comprise the fragment of the continuous and appointment that refers to polynucleotide sequence, wherein polynucleotide sequence can compare with reference sequences and wherein this polynucleotide sequence part in this comparison window can comprise and add or disappearance (being the room) than reference sequences (do not comprise and add or disappearance) so that the best of two sequences comparison.Usually, comparison window length is at least 20 continuous Nucleotide, chooses wantonly and can be 30,40,50,100 or longer.Those skilled in the art recognize that, owing in polynucleotide sequence, add due to the room and high similarity reference sequences, usually introduce gap penalty and from coupling number deduction gap penalty for avoiding.
The whole bag of tricks that Nucleotide and aminoacid sequence are compared to make comparisons is well known in the art.Local homology's algorithm (BESTFIT) (Smith and Waterman, (1981) Adv.Appl.Math2:482 (Smith and Waterman,, " applied mathematics progress " in 1981, the 2nd volume, the 482nd page)) can carry out the best comparison to being used for sequence relatively; Also can adopt homology contrast algorithm (GAP) (Needleman and Wunsch, (1970) J.Mol.Biol.48:443-53 (Needleman and Wunsch,, " molecular biology magazine " in 1970, the 48th volume, the 443-453 page or leaf)); Similarity searching method (Tfasta and Fasta) (Pearson and Lipman, (1988) Proc.Natl.Acad.Sci.USA 85:2444 (Pearson and Lipman,, " institute of NAS periodical " in 1988, the 85th volume, the 2444th page)); The computerize of these algorithms is implemented to include, but are not limited to: CLUSTAL, Wisconsin Genetics Software in the PC/Gene program of Intelligenetics (mountain scene city, California (Mountain View, California))
Figure BDA00002091896500241
The 8th edition (can derive from Genetics Computer Group (
Figure BDA00002091896500242
GAP, BESTFIT, BLAST, FASTA and TFASTA in the program (Accelrys company (San Diego, California (San Diego, CA))).The CLUSTAL program is by describing in detail such as Publication about Document: Higgins and Sharp, (1988) Gene 73:237-44 (Higgins and Sharp,, " gene ", the 73rd volume, 237-244 page or leaf in 1988); Higgins and Sharp, (1989) CABIOS 5:151-3 (Higgins and Sharp,, " application of computer in bio-science ", the 5th volume, 151-153 page or leaf in 1989); Corpet, et al., (1988) Nucleic Acids Res.16:10881-90 (people such as Corpet,, " nucleic acids research ", the 16th volume, 10881-10890 page or leaf in 1988); Huang, et a1., (1992) Computer Applications in the Biosciences 8:155-65 (people such as Huang, 1992, " application of computer in bio-science ", the 8th volume, the 155-165 page or leaf) and Pearson, et al., (1994) Meth.Mol.Biol.24:307-31 (people such as Pearson, " molecular biology method ", the 24th volume, 307-331 page or leaf).The preferable procedure that is used for the best overall comparison of a plurality of sequences is PileUp (Feng and Doolittle, (1987) J.Mol.Evol., 25:351-60 (Feng and Doolittle, 1987, " molecular evolution magazine ", the 25th volume, the 351-360 page or leaf), it is similar to Higgins and Sharp, (1989) CABIOS 5:151-53 (Higgins and Sharp, 1989, " application of computer in bio-science ", the 5th volume, 151-153 page or leaf) method of describing, and incorporate by reference accordingly this paper into).The BLAST family program that can be used for database similarity search comprises: BLASTN is used for the nucleotide query sequence and inquires about for the RiboaptDB sequence; BLASTX is used for the nucleotide query sequence and inquires about for the Protein Data Bank sequence; BLASTP is used for the protein search sequence and inquires about for the Protein Data Bank sequence; TBLASTN is used for the protein search sequence and inquires about for the RiboaptDB sequence; And TBLASTX, be used for the nucleotide query sequence and inquire about for the RiboaptDB sequence.Referring to CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Chapter 19, Ausubel, et al., eds., Greene Publishing and Wiley-Interscience, New York (1995) (" up-to-date experimental methods of molecular biology compilation ", the 19th chapter, the people such as Ausubel edit, Green press and Wiley-Interscience, New York, nineteen ninety-five).
GAP utilizes the algorithm of Needleman and Wunsch (ibid) to seek the comparison of two complete sequence, and this comparison makes coupling number maximum and makes the room number minimum.GAP can consider all possible comparison and null position, and produces the comparison in coupling base with maximum number and minimum room.It allows to provide to mate room generation point penalty and the room extension point penalty that the base number is unit.GAP must utilize the room of coupling to produce the point penalty number for each room of its insertion.If select to extend point penalty greater than zero room, GAP must utilize room length to multiply by room extension point penalty for the room of each insertion in addition.Wisconsin Genetics Software
Figure BDA00002091896500251
Acquiescence room in the 10th edition produces the point penalty value and room extension point penalty value is respectively 8 and 2.The room produces and room extension point penalty can represent with the integer that is selected from 0-100.Thereby for example, the room produces and room extension point penalty can be 0,1,2,3,4,5,6,7,8,9,10,15,20,30,40,50 or larger.
GAP provides a member in the family with best comparison.May there be many members in this family, but other members do not have better quality.GAP shows four figure of merits that are used for comparison: quality, ratio, identity and similarity.Quality is the index (metric) that is maximized for aligned sequences.Ratio is that quality is divided by the base number in the shorter section.Identity percentage ratio is the percentage ratio of the symbol of actual match.Similarity percentage ratio is the percentage ratio of similar symbol.To ignore corresponding to the symbol in room.When the marking matrix value of pair of symbols during more than or equal to 0.50 (similarity threshold value), similarity is given a mark.Wisconsin Genetics Software
Figure BDA00002091896500261
Used marking matrix is that BLOSUM62 is (referring to Henikoff and Henikoff in the 10th edition, (1989) Proc.Natl.Acad.Sci.USA89:10915 (Henikoff and Henikoff, 1989, " institute of NAS periodical ", the 89th volume, the 10915th page)).
Except as otherwise noted, otherwise sequence identity/similarity that this paper provides refers to use the BLAST2.0 routine package, value (the Altschul that adopts default parameters to obtain, et al., (1997) Nucleic Acids Res.25:3389-402 (people such as Altschul,, " nucleic acids research " in 1997, the 25th volume, the 3389-3402 page or leaf)).
As one of ordinary skill will be understood, blast search putative protein matter can the stochastic sequence modeling.Yet many true protein comprise nonrandom sequence area, and this nonrandom sequence can be with poly-section, short period and repeats or be rich in one or more amino acid whose zones.This low-complexity zone can be compared between incoherent protein, although other zones of this protein are fully dissimilar.Can adopt multiple low-complexity filter to reduce this low-complexity comparison.For example, can use separately or unite use SEG (Wooten and Federhen, (1993) Comput.Chem.17:149-63 (Wooten and Federhen, 1993, " calculational chemistry magazine ", the 17th volume, and XNU (Claverie and States the 149-163 page or leaf)), (1993) Comput.Chem.17:191-201 (Claverie and States, 1993, " calculational chemistry magazine ", the 17th volume, 191-201 page or leaf)) the low-complexity filter.
In the situation of two polynucleotide or peptide sequence, " sequence identity " used herein or " identity " refer to when compare to obtain in the comparison window of appointment maximum to seasonable two sequences in identical residue.When sequence identity percentage ratio uses for protein, will be appreciated that, not identical residue position often difference is conservative amino acid replacement, wherein amino-acid residue is had the radical amino acid replacement of similar chemical property (for example electric charge or hydrophobicity) by other, therefore can not change the functional property of molecule.Be conservative substitution such as the infructescence difference, then can raise per-cent sequence identity to proofread and correct the conservative character of displacement.Difference is that the sequence of this conservative substitution is said to be and has " sequence similarity " or " similarity ".The method of making this adjusting is well-known to those skilled in the art.Usually, this relates to conservative substitution marking is part mispairing rather than fully mispairing, thereby improves sequence identity percentage ratio.Thereby for example, if identical amino acid gives 1 minute, non-conservative displacement gives 0 minute, and then conservative substitution gives the mark between 0 to 1.For example, according to Meyers and Miller, (1988) Computer Applic.Biol.Sci.4:11-17 (Meyers and Miller, 1988, " application of computer in bio-science ", the 4th volume, the 11-17 page or leaf) algorithm calculates the mark of conservative substitution, for example as in program PC/GENE (Intelligenetics (California, USA mountain scene city (Mountain View, California, USA))), realize.
As used herein, " sequence identity percentage ratio " means by compare the determined numerical value of sequence of two best comparisons in comparison window, wherein the part of polynucleotide sequence in comparison window compared to comprise with reference sequences (do not comprise and add or disappearance) and added or disappearance (being the room), so that the best of two sequences comparison.This percentage ratio is to calculate like this: determine to occur the number of position of identical nucleic acid base or amino-acid residue with the number of the position that obtains mating in two sequences, the number of the position of coupling divided by the overall number of the position in the comparison window, then be multiply by the result 100 to obtain sequence identity percentage ratio.
When " substantially the same " of term polynucleotide sequence means to utilize one of described comparison program to adopt canonical parameter and reference sequences relatively, polynucleotide comprise the sequence identity that has between the 50-100%, preferred at least 50% sequence identity, preferred at least 60% sequence identity, preferably at least 70%, more preferably at least 80%, the more preferably sequence of at least 90% and most preferably at least 95% sequence identity.The technician will recognize that, can these be worth the corresponding identity with the coded protein of definite two nucleotide sequences by consideration codon degeneracy, amino acid similarity, reading frame location etc. suitable adjusting.The substantially the same sequence identity that usually means between the 55-100% of aminoacid sequence that is used for these purposes, preferably at least 55%, preferably at least 60%, more preferably at least 70%, 80%, 90%, most preferably at least 95%.
Another substantially the same indication of nucleotide sequence is whether two molecules hybridize under stringent condition each other.The degeneracy of genetic code allows many amino-acid substitutions, and this amino-acid substitution causes variation in the nucleotide sequence of coding same amino acid, thereby might this dna sequence dna codified phase homopolypeptide but each other hybridization under stringent condition.For example, when a nucleic acid copy of the maximum codon degeneracy generation that utilizes genetic code to allow, this may occur.Article two, nucleotide sequence is that a substantially the same indication is, the polypeptide generation immunological cross-reaction that the polypeptide of the first nucleic acid encoding and the second nucleic acid are coded.
In the situation of peptide, term " substantially the same " refers to that peptide is included in and specifies on the comparison window and reference sequences has sequence identity between the 55-100%; Preferably have at least 55% sequence identity with reference sequences, preferred 60%, preferred 70%, more preferably 80%, at least 90% or 95% sequence identity most preferably.Preferably, utilize the homology alignment algorithm of Needleman and Wunsch (ibid) to carry out the best comparison.Article two, peptide sequence is that substantially the same indication is, a kind of peptide can with the antibody generation immune response that produces for the second peptide.Thereby for example, if certain peptide and the second peptide difference only are conservative substitution, then these two kinds of peptides are substantially the same.In addition, when certain peptide and the second peptide difference were non-conservative variation, if the epi-position of antibody recognition is substantially the same, then they were substantially the same." basically similar " peptide has aforesaid sequence, exception be that not identical residue position difference can be that conserved amino acid changes.
The invention discloses round the clock polynucleotide and polypeptide.Nucleus thuja acid of the present invention and protein have show their regulating cell quantity and thereby the expression pattern that plays an important role in development of plants.These polynucleotide are expressed in the various plants tissue.These polynucleotide and polypeptide thereby provide and handle the chance that development of plants changes seed and nutritive issue growth, arrangement of time or composition.This can be used for producing sterile plant, without the seed plant or have the plant that the endosperm of change forms.
Whether follow species formation pattern by the protein relation that mutual blast search and assessment are inferred, identify the corn ortholog gene of Arabidopis thaliana and paddy rice diel rhythm gene, the mode of oscillation of then inquiring about leaf and fringe tissue.Adopt these standards, in the corn homologous gene, identified several main core components, comprise CCA1/LHY, TOC1, PRR7/3, GI and ZTL (Fig. 1).
Two TOC1 homologous gene ZmTOCa and ZmTOCb have been identified in this research, and they are positioned respectively No. 5 and No. 4 karyomit(e)s.Transcribing all of two genes reaches peak value at 6pm, and this conforms to Arabidopis thaliana TOC1 genetic expression.TOC1 is the member that puppet is replied regulatory factor (PRR) family, this family is by upper five the conservative PRR genomic constitution (Murakami that evolve in Arabidopis thaliana and the paddy rice, et al., (2007) the Biosci Biotechnol Biochem 71:1107-1110 (people such as Murakami, 2007, " bio-science, biotechnology and biological chemistry ", the 71st volume, 1107-1110 page or leaf); Murakami, et al., (2003) Plant Cell Physiol 44:1229-1236 (the village upper class,, " plant cell physiology ", the 44th volume, 1229-1236 page or leaf in 2003)).Except two ZmTOC1 homologous genes, ZmPRR73, ZmPRR37 and ZmPRR59 have also been identified in this research, and they are based on the sequence similarity level according to (the village upper class, (2003)) of paddy rice PRR unnamed gene.Two ZEITLUPE homologous genes (Kim, et al., (2007) Nature 449:356-360 (people such as Kim have also been identified, 2007, " nature ", the 449th volume, the 356-360 page or leaf)) ZmZTLa and ZmZTLb, they are positioned No. 2 and No. 4 karyomit(e)s.Two corn ortholog gene gigz1A of GIGANTI and gigz1B be the existing (Miller that describes before this, et al., (2008) the Planta 227:1377-1388 (people such as Miller, 2008, " plant ", the 227th volume, the 1377-1388 page or leaf)), confirmed their vibrations in fringe and leaf at this.Analyze the circulation (Fig. 2) of further having confirmed core component ZmCCA, ZmLHY, ZmTOC1a and ZmTOC1b by RT-PCR.Compare with leaf texture, the amplitude of the core constituent element in the growth period fringe weakens, but still strong.These data show that majority of plant core oscillator system for example organizes at non-photosynthesis and work in the fringe, but vibrator output obviously separates far away with the transcriptional machinery that affect downstream Circadian Expression change.
Determined most of function that the transcript of round the clock regulation and control spreads all over the leaf of Semen Maydis cell.This paper determines that 6674 transcripts can being regulated and control round the clock (come from 10,037 Agilent (Agilent) array probe) representative surpasses whole transcripts of 22% the expression that detects, and these 6674 transcripts can belong to 1716 different gene ontologies (GO) and 22 KOG functional categories.
Generally speaking, each gene peak only has a peak in its diurnal cycle.When these genes being belonged to each function items and draw the relative enrichment of these function items in one day scope, most of function has the significant enrichment of peculiar pattern of time in a day.Yet some function items also obviously trend towards having bimodal pattern, wherein at 10AM peak in the middle of the morning are arranged, between the lights 6PM or evening 10PM second peak arranged.Surpass 18% function items and be classified as bimodal regulation and control, and according to the morning or afternoon the peak relative enrichment further segment.Add each function of only being appointed as in one day at place, peak peaking, 94.5% of described 1738 functions belong to a kind of in this two-mode, and only remaining 95 belong to " other " pattern.
The function items of common bimodal pattern represents widely gene enrichment function classification, for example protein kinase activity, signal transduction mechanism or amino acid transport and metabolism.(Fig. 5) therefore, these bimodal patterns often also have adequate representation in all day, are not 10AM and 6/10PM.Yet gene and function enrichment peak usually occur also between the lights in the middle of the morning/again occur evening, and this remains the round the clock principal character of pattern.In this experiment, sunrise is 6:02AM, and day is dropped on 8:40PM.Therefore, sunrise is before the function peak of 10AM 4 hours, but sunset is after the 6PM time point before 2.45 hours and the 10PM time point 1.25 hours.Additional time point can provide higher resolving power, but the function enrichment index of 10AM＞6/10PM pattern exceeds 70% than 6PM＞10AM pattern in bimodal pattern, and this may be relevant with respect to the mal-distribution of sunrise and sunset with these time points.Perhaps, some functional categories may inherently in enrichment, reflect potential biological trend in the stage in the morning.
1643 or 94.5% function items belong to a time peak pattern, and this shows that each function had the evolution of quite determining in one day.As seen functional group is not to be uniformly distributed in one day different steps, but shows specific pattern and deflection.The functional category of enrichment at dawn for example comprises: to response, lipolysis metabolism and the hormone signal conduction of cold.Then in the middle of the morning, a plurality of hormone response functions are by enrichment.Expect that synthesized by photosynthesizer I and II, chlorophyll noon and the monodehydroascorbate reductase (MDAR) of participation antioxidant generation is leading.At dusk and demonstrate the enrichment of obvious rrna and dna damage reparation (comprising helicase, Telomerase and endonuclease activity) evening, illustrate that karyomit(e) and rrna repair system are activated.In addition, sucrose transhipment and pentosephosphate shunt between the lights/evening peaking, the dynamics variation of chloroplast(id) carbohydrate metabolism has been described.The late into the night, the peak comprised ruddiness:: the far-red light conduction, as described in foreword, it not only regulates and control core clock, and the regulation and control Hydrogen Peroxide Metabolism.Night usually caspase (sample) activity, Photosystem I I katabolism, Nucleotide transhipment and metabolism and the acetyl-CoA combined function relevant with necrocytosis all reach peak value.Other peak patterns irregular but that attract people's attention are the amino acid glycosylations that all reaches peak value at 6PM and 2AM, and the malic enzyme and the calmodulin combination that reach peak value at 10AM and 2AM.These just describe several examples of the very complicated situation of complete stool plant cell physiology.
Although it should be noted that many genes and function are regulated and control round the clock, the member that most of functional category only has minority to be regulated and control round the clock.In 1738 functional categories, average coverage rate is 28.2%, and median is 20%, and mode is about 15%.The transcript of regulation and control can't represent the functional category that comprises a plurality of genes fully round the clock, for the transcript of round the clock regulation and control, only has seldom significantly enrichment of functional category.In round the clock regulation and control group, GO:0004614 phosphoglucomutase activity has 5 in 6 transcripts, and the GO:0009926 Polar Transport of Auxin has 3 in 4 transcripts.These are found the transcript participation of round the clock regulation and control of explanation but do not dominate these different functions.
In analysis, identified the gene that a plurality of corns are regulated and control round the clock.471 sequences altogether, comprise from the sequence of prematurity fringe, in leaf texture, have high amplitude/significantly circulation sequence and with NUE and carbon:: the multiple sequence that the nitrogen function is relevant.These sequences comprise polypeptide and the relative promotor of ORF, coding.
Nucleic acid
The present invention provides especially and comprises round the clock RNA, the DNA of polynucleotide and the nucleic acid of their analogue and/or chimeric separation.
The present invention also is included as in different organisms and expresses and polynucleotide through optimizing.For example, for the expression of polynucleotide in maize plant, variable this sequence is to solve specific codon preference and to change GC content, as described in according to the people such as Murray (ibid).Use about the corn codon from 28 kinds of genes of maize plant and in the table 4 of the people such as Murray (ibid), to list.
Round the clock nucleic acid of the present invention comprises the round the clock polynucleotide of separation, and described polynucleotide comprise:
(a) encode polypeptide round the clock and through the polynucleotide of conservative variant that modify and polymorphism;
(b) with (a) or the polynucleotide of polynucleotide (b) with at least 70% sequence identity;
(c) (a) or the complementary sequence of polynucleotide (b).
Following table 1 has been listed the concrete composition of polynucleotide disclosed herein and polypeptide
Table 1
Title Plant species Polynucleotide/polypeptide SEQ ID NO：
ZmTOC1b Corn (Zea mays) Polynucleotide SEQ ID NO：1
ZmMYB.L Corn (Zea mays) Polynucleotide SEQ ID NO：2
ZmZTLa Corn (Zea mays) Polynucleotide SEQ ID NO：3
ZmZTLb Corn (Zea mays) Polynucleotide SEQ ID NO：4
ZmPRR37 Corn (Zea mays) Polynucleotide SEQ ID NO：5
ZmPRR59 Corn (Zea mays) Polynucleotide SEQ ID NO：6
The ZmCO analogue Corn (Zea mays) Polynucleotide SEQ ID NO：7
The ZmCCA1 genome Corn (Zea mays) Polynucleotide SEQ ID NO：8
The ZmCCA1 exons 1 Corn (Zea mays) Polynucleotide SEQ ID NO：9
The ZmCCA1 exon 2 Corn (Zea mays) Polynucleotide SEQ ID NO：10
The ZmCCA1 exon 3 Corn (Zea mays) Polynucleotide SEQ ID NO：11
ZmCCA1 exon 4 Corn (Zea mays) Polynucleotide SEQ ID NO：12
ZmCCA1 exon 5 Corn (Zea mays) Polynucleotide SEQ ID NO：13
The ZmCCA1 exon 6 Corn (Zea mays) Polynucleotide SEQ ID NO：14
The ZmCCA1 exon 7 Corn (Zea mays) Polynucleotide SEQ ID NO：15
ZmCCA1 exon 8 Corn (Zea mays) Polynucleotide SEQ ID NO：16
ZmCCA1 exon 9 Corn (Zea mays) Polynucleotide SEQ ID NO：17
ZmCCA1 exons 10 Corn (Zea mays) Polynucleotide SEQ ID NO：18
ZmCCA1 exons 11 Corn (Zea mays) Polynucleotide SEQ ID NO：19
The ZmLHY genome Corn (Zea mays) Polynucleotide SEQ ID NO：20
The ZmLHY exons 1 Corn (Zea mays) Polynucleotide SEQ ID NO：21
The ZmLHY exon 2 Corn (Zea mays) Polynucleotide SEQ ID NO：22
The ZmLHY exon 3 Corn (Zea mays) Polynucleotide SEQ ID NO：23
ZmLHY exon 4 Corn (Zea mays) Polynucleotide SEQ ID NO：24
ZmLHY exon 5 Corn (Zea mays) Polynucleotide SEQ ID NO：25
The ZmLHY exon 6 Corn (Zea mays) Polynucleotide SEQ ID NO：26
The ZmLHY exon 7 Corn (Zea mays) Polynucleotide SEQ ID NO：27
ZmLHY exon 8 Corn (Zea mays) Polynucleotide SEQ ID NO：28
ZmLHY exon 9 Corn (Zea mays) Polynucleotide SEQ ID NO：29
ZmLHY exons 10 Corn (Zea mays) Polynucleotide SEQ ID NO：30
Promotor #1 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：31
Promotor #2 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：32
Promotor #3 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：33
Promotor #4 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：34
The ZmCCA1 promotor Corn (Zea mays) Polynucleotide SEQ ID NO：35
The ZmLHY promotor Corn (Zea mays) Polynucleotide SEQ ID NO：36
Promotor #7 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：37
Promotor #8 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：38
Promotor #9 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：39
The ZmTOCa promotor Corn (Zea mays) Polynucleotide SEQ ID NO：40
The fringe promotor 1 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：41
The fringe promotor 2 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：42
The fringe promotor 3 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：43
The fringe promotor 4 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：44
The fringe promotor 5 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：45
The fringe promotor 7 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：46
The fringe promotor 8 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：47
The fringe promotor 9 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：48
The fringe promotor 10 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：49
The fringe promotor 11 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：50
The fringe promotor 12 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：51
The fringe promotor 13 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：52
The fringe promotor 14 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：53
The fringe promotor 15 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：54
The fringe promotor 16 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：55
The NUE promotor 1 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：56
The NUE promotor 2 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：57
The NUE promotor 3 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：58
The NUE promotor 4 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：59
The NUE promotor 5 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：60
The NUE promotor 6 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：61
The NUE promotor 7 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：62
The NUE promotor 8 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：63
The NUE promotor 9 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：64
The NUE promotor 10 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：65
The NUE promotor 11 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：66
The NUE promotor 12 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：67
The NUE promotor 13 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：68
The NUE promotor 14 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：69
The NUE promotor 15 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：70
The NUE promotor 16 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：71
The NUE promotor 17 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：72
The NUE promotor 18 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：73
The NUE promotor 19 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：74
The NUE promotor 20 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：75
The NUE promotor 21 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：76
The NUE promotor 22 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：77
The NUE promotor 23 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：78
The NUE promotor 24 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：79
The NUE promotor 25 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：80
The NUE promotor 26 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：81
The NUE promotor 27 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：82
The NUE promotor 28 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：83
The NUE promotor 29 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：84
The NUE promotor 30 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：85
The NUE promotor 31 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：86
The NUE promotor 32 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：87
The NUE promotor 33 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：88
The NUE promotor 34 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：89
The NUE promotor 35 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：90
The NUE promotor 36 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：91
The NUE promotor 37 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：92
The NUE promotor 38 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：93
The NUE promotor 39 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：94
The NUE promotor 40 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：95
The NUE promotor 41 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：96
The NUE promotor 42 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：97
The NUE promotor 43 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：98
The NUE promotor 44 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：99
The NUE promotor 45 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：100
The NUE promotor 46 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：101
The NUE promotor 47 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：102
The NUE promotor 48 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：103
The NUE promotor 49 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：104
The NUE promotor 50 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：105
The NUE promotor 51 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：106
The NUE promotor 52 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：107
The NUE promotor 53 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：108
The NUE promotor 54 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：109
The NUE promotor 55 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：110
The NUE promotor 56 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：111
The NUE promotor 57 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：112
The NUE promotor 58 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：113
The NUE promotor 59 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：114
The NUE promotor 60 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：115
The NUE promotor 61 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：116
The AMP promotor 1 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：117
The AMP promotor 2 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：118
The AMP promotor 3 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：119
The AMP promotor 4 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：120
The AMP promotor 5 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：121
The AMP promotor 6 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：122
The AMP promotor 7 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：123
The AMP promotor 8 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：124
The AMP promotor 9 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：125
The AMP promotor 10 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：126
The AMP promotor 11 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：127
The AMP promotor 12 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：128
The AMP promotor 13 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：129
The AMP promotor 14 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：130
The AMP promotor 15 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：131
The AMP promotor 16 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：132
The AMP promotor 17 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：133
The AMP promotor 18 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：134
The AMP promotor 19 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：135
The AMP promotor 20 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：136
The AMP promotor 21 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：137
The AMP promotor 22 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：138
The AMP promotor 23 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：139
The AMP promotor 24 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：140
The AMP promotor 25 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：141
The AMP promotor 26 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：142
The AMP promotor 27 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：143
The AMP promotor 28 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：144
The AMP promotor 29 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：145
The AMP promotor 30 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：146
The AMP promotor 31 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：147
The AMP promotor 32 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：148
The AMP promotor 33 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：149
The AMP promotor 34 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：150
The AMP promotor 35 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：151
The AMP promotor 36 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：152
The AMP promotor 37 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：153
The AMP promotor 38 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：154
The AMP promotor 39 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：155
The AMP promotor 40 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：156
The AMP promotor 41 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：157
The AMP promotor 42 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：158
The AMP promotor 43 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：159
The AMP promotor 44 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：160
The AMP promotor 45 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：161
The AMP promotor 46 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：162
The AMP promotor 47 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：163
The AMP promotor 48 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：164
The AMP promotor 49 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：165
The AMP promotor 50 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：166
The AMP promotor 51 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：167
The AMP promotor 52 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：168
The AMP promotor 53 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：169
The AMP promotor 54 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：170
The AMP promotor 55 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：171
The AMP promotor 56 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：172
The AMP promotor 57 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：173
The AMP promotor 58 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：174
The AMP promotor 59 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：175
The AMP promotor 60 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：176
The AMP promotor 61 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：177
The AMP promotor 62 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：178
The AMP promotor 63 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：179
The AMP promotor 64 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：180
The AMP promotor 65 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：181
The AMP promotor 66 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：182
The AMP promotor 67 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：183
Fringe 1 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：184
Fringe 1 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：185
Fringe 2 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：186
Fringe 2 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：187
Fringe 3 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：188
Fringe 3 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：189
Fringe 4 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：190
Fringe 4 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：191
Fringe 5 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：192
Fringe 5 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：193
Fringe 6 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：194
Fringe 6 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：195
Fringe 7 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：196
Fringe 7 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：197
Fringe 8 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：198
Fringe 8 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：199
Fringe 9 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：200
Fringe 9 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：201
Fringe 10 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：202
Fringe 10 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：203
Fringe 11 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：204
Fringe 11 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：205
Fringe 12 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：206
Fringe 12 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：207
Fringe 13 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：208
Fringe 13 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：209
Fringe 14 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：210
Fringe 14 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：211
Fringe 15 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：212
Fringe 15 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：213
Fringe 16 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：214
Fringe 16 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：215
NUE 1 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：216
NUE 1 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：217
NUE 2 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：218
NUE 2 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：219
NUE 3 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：220
NUE 3 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：221
NUE 4 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：222
NUE 4 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：223
NUE 5 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：224
NUE 5 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：225
NUE 6 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：226
NUE 6 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：227
NUE 7 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：228
NUE 7 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：229
NUE 8 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：230
NUE 8 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：231
NUE 9 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：232
NUE 9 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：233
NUE 10 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：234
NUE 10 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：235
NUE 11 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：236
NUE 11 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：237
NUE 12 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：238
NUE 12 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：239
NUE 13 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：240
NUE 13 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：241
NUE 14 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：242
NUE 14 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：243
NUE 15 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：244
NUE 15 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：245
NUE 16 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：246
NUE 16 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：247
NUE 17 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：248
NUE 17 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：249
NUE 18 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：250
NUE 18 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：251
NUE 19 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：252
NUE 19 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：253
NUE 20 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：254
NUE 20 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：255
NUE 21 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：256
NUE 21 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：257
NUE 22 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：258
NUE 22 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：259
NUE 23 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：260
NUE 23 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：261
NUE 24 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：262
NUE 24 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：263
NUE 25 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：264
NUE 25 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：265
NUE 26 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：266
NUE 26 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：267
NUE 27 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：268
NUE 27 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：269
NUE 28 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：270
NUE 28 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：271
NUE 29 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：272
NUE 29 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：273
NUE 30 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：274
NUE 30 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：275
NUE 31 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：276
NUE 31 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：277
NUE 32 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：278
NUE 32 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：279
NUE 33 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：280
NUE 33 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：281
NUE 34 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：282
NUE 34 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：283
NUE 35 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：284
NUE 35 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：285
NUE 36 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：286
NUE 36 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：287
NUE 37 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：288
NUE 37 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：289
NUE 38 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：290
NUE 38 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：291
NUE 39 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：292
NUE 39 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：293
NUE 40 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：294
NUE 40 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：295
NUE 41 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：296
NUE 41 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：297
NUE 42 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：298
NUE 42 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：299
NUE 43 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：300
NUE 43 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：301
NUE 44 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：302
NUE 44 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：303
NUE 45 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：304
NUE 45 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：305
NUE 46 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：306
NUE 46 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：307
NUE 47 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：308
NUE 47 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：309
NUE 48 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：310
NUE 48 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：311
NUE 49 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：312
NUE 49 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：313
NUE 50 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：314
NUE 50 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：315
NUE 51 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：316
NUE 51 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：317
NUE 52 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：318
NUE 52 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：319
NUE 53 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：320
NUE 53 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：321
NUE 54 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：322
NUE 54 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：323
NUE 55 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：324
NUE 55 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：325
NUE 56 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：326
NUE 56 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：327
NUE 57 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：328
NUE 57 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：329
NUE 58 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：330
NUE 58 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：331
NUE 59 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：332
NUE 59 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：333
NUE 60 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：334
NUE 60 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：335
NUE 61 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：336
NUE 61 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：337
AMP 1 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：338
AMP 1 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：339
AMP 2 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：340
AMP 2 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：341
AMP 3 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：342
AMP 3 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：343
AMP 4 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：344
AMP 4 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：345
AMP 5 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：346
AMP 5 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：347
AMP 6 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：348
AMP 6 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：349
AMP 7 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：350
AMP 7 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：351
AMP 8 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：352
AMP 8 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：353
AMP 9 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：354
AMP 9 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：355
AMP 10 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：356
AMP 10 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：357
AMP 11 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：358
AMP 11 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：359
AMP 12 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：360
AMP 12 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：361
AMP 13 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：362
AMP 13 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：363
AMP 14 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：364
AMP 14 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：365
AMP 15 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：366
AMP 15 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：367
AMP 16 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：368
AMP 16 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：369
AMP 17 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：370
AMP 17 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：371
AMP 18 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：372
AMP 18 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：373
AMP 19 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：374
AMP 19 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：375
AMP 20 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：376
AMP 20 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：377
AMP 21 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：378
AMP 21 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：379
AMP 22 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：380
AMP 22 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：381
AMP 23 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：382
AMP 23 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：383
AMP 24 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：384
AMP 24 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：385
AMP 25 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：386
AMP 25 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：387
AMP 26 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：388
AMP 26 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：389
AMP 27 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：390
AMP 27 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：391
AMP 28 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：392
AMP 28 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：393
AMP 29 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：394
AMP 29 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：395
AMP 30 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：396
AMP 30 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：397
AMP 31 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：398
AMP 31 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：399
AMP 32 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：400
AMP 32 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：401
AMP 33 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：402
AMP 33 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：403
AMP 34 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：404
AMP 34 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：405
AMP 35 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：406
AMP 35 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：407
AMP 36 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：408
AMP 36 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：409
AMP 37 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：410
AMP 37 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：411
AMP 38 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：412
AMP 38 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：413
AMP 39 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：414
AMP 39 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：415
AMP 40 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：416
AMP 40 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：417
AMP 41 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：418
AMP 41 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：419
AMP 42 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：420
AMP 42 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：421
AMP 43 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：422
AMP 43 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：423
AMP 44 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：424
AMP 44 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：425
AMP 45 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：426
AMP 45 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：427
AMP 46 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：428
AMP 46 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：429
AMP 47 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：430
AMP 47 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：431
AMP 48 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：432
AMP 48 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：433
AMP 49 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：434
AMP 49 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：435
AMP 50 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：436
AMP 50 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：437
AMP 51 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：438
AMP 51 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：439
AMP 52 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：440
AMP 52 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：441
AMP 53 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：442
AMP 53 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：443
AMP 54 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：444
AMP 54 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：445
AMP 55 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：446
AMP 55 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：447
AMP 56 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：448
AMP 56 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：449
AMP 57 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：450
AMP 57 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：451
AMP 58 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：452
AMP 58 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：453
AMP 59 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：454
AMP 59 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：455
AMP 60 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：456
AMP 60 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：457
AMP 61 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：458
AMP 61 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：459
AMP 62 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：460
AMP 62 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：461
AMP 63 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：462
AMP 63 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：463
AMP 64 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：464
AMP 64 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：465
AMP 65 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：466
AMP 65 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：467
AMP 66 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：468
AMP 66 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：469
AMP 67 round the clock Corn (Zea mays) Polynucleotide SEQ ID NO：470
AMP 67 round the clock Corn (Zea mays) Polypeptide SEQ ID NO：471
The structure of nucleic acid
The nucleic acid of the separation among the present invention can be standby in order to the below legal system: (a) standard recombination method, (b) synthetic technology, or its combination.In certain embodiments, polynucleotide of the present invention will or otherwise make up from fungi or bacterial clone, amplification.
Described nucleic acid can comprise the sequence except polynucleotide of the present invention expediently.For example, the multiple clone site that comprises one or more endonuclease restriction sites can be inserted in the nucleic acid to help to separate these polynucleotide.In addition, but can insert the polynucleotide of the present invention that translation sequences has been translated to help separation.For example, the six histidine mark sequences means of providing convenience are come purifying protein of the present invention.Nucleic acid of the present invention (getting rid of described polynucleotide sequence) randomly is to be used for the clone of polynucleotide of the present invention and/or carrier, adapter or the joint of expression.Other sequence can be added into this clone and/or expressed sequence and optimize them the clone and/or the function in expressing, to help the described polynucleotide of separation, or improve described polynucleotide to intracellular introducing.Usually, the length that the length of nucleic acid of the present invention deducts its polynucleotide of the present invention is for less than 20 kilobase pair, often less than 15kb, usually less than 10kb.The use of cloning vector, expression vector, adapter and joint is known in the art.Exemplary nucleic acid comprises such as following carrier: M13, λ ZAP Express, λ ZAP II, λ gt10, λ gt11, pBK-CMV, pBK-RSV, pBluescript II, λ DASH II, λ EMBL 3, λ EMBL 4, pWE15, SuperCos 1, SurfZap, Uni-ZAP, pBC, pBS+/-, pSG5, pBK, pCR-Script, pET, pSPUTK, p3 ' SS, pGEM, pSK+/-, pGEX, pSPORTI and II, pOPRSVI CAT, pOPI3 CAT, pXT1, pSG5, pPbac, pMbac, pMC1neo, pOG44, pOG45, pFRT β GAL, pNEO β GAL, pRS403, pRS404, pRS405, pRS406, pRS413, pRS414, pRS415, pRS416, λ MOSSlox and λ MOSElox.Choose wantonly for carrier of the present invention and include but not limited to λ ZAP II and pGEX.The description of relevant various nucleic acid, referring to for example Stratagene Cloning Systems, catalog number (Cat.No.) 1995,1996,1997 (California La Jolla (La Jolla, CA)) and peace agate West Asia bio tech ltd (Amersham Life Sciences, Inc), catalog number (Cat.No.) ' 97 (highland, Arlington, Illinois (Arlington Heights, IL)).
Make up the synthetic method of nucleic acid
Isolated nucleic acid among the present invention also can be by direct chemosynthesis preparation, for example use phosphotriester method (Narang, et al., (1979) Meth.Enzymol.68:90-9 (people such as Narang, 1979, " Enzymology method ", the 68th volume, the 90-99 page or leaf)), phosphodiester method (Brown, et al., (1979) Meth.Enzymol.68:109-51 (people such as Brown, 1979, " Enzymology method ", the 68th volume, the 109-151 page or leaf)), diethyl phosphoramidite method (Beaucage et al., (1981) Tetra.Letts.22 (20): the 1859-62 (people such as Beaucage, 1981, " tetrahedron communication ", the 20th phase of the 22nd volume, 1859-1862 page or leaf)), the described solid phase phosphoramidite three ester methods of the people such as Beaucage as above, with for example such as Needham-VanDevanter, et al., the described automatic DNA synthesizer DNA of (1984) Nucleic Acids Res.12:6159-68, and U.S. Patent No. 4,458,066 solid phase support method.Chemosynthesis produces single stranded oligonucleotide usually.This can be by changing double-stranded DNA into complementary sequence hybridization or by this strand is carried out polymerization as template with archaeal dna polymerase.The technician will recognize that, although the chemosynthesis of DNA is confined to the sequence of about 100 bases, and the sequence that can obtain to grow by connecting short sequence.
UTR and codon preference
Generally speaking, found that translation efficiency is subjected to 5 ' non-coding region or the non-translational region (regulation and control of the particular sequence element in 5 ' UTR) of RNA.Positive sequence motifs comprises translation initiation consensus sequence (Kozak, (1987) Nucleic Acids Res.15:8125 (Kozak,, " nucleic acids research " in 1987, the 15th volume, the 8125th page)) and 5＜G 7 methyl GpppG RNA cap structures (Drummond, et al., (1985) Nucleic Acids Res.13:7375 (people such as Drummond, 1985, " nucleic acids research ", the 13rd volume, the 7375th page)).Negative element comprises 5 ' UTR stem-ring structure (Muesing in the stable molecule, et al., (1987) the Cell 48:691 (people such as Muesing, 1987, " cell ", the 48th volume, the 691st page)) and 5 ' UTR in AUG sequence or front short open reading frame (Kozak (ibid), Rao that suitable AUG is arranged, et al., (1988) Mol.and Cell.Biol.8:284 (people such as Rao,, " molecule and cytobiology " in 1988, the 8th volume, the 284th page)).Therefore, the invention provides for 5 of the translation of regulating allogeneic coding sequence ' and/or 3 ' UTR district.
In addition, the peptide coding section that can modify polynucleotide of the present invention uses to change codon.Can to adopt altered codon to use, to change expression or the codon use in the optimization heterologous sequence in order in corn, expressing in required host of translation efficiency and/or Optimized Coding Based sequence.Codon in the coding region of polynucleotide of the present invention uses the software package (as deriving from University of Wisconsin Genetics Computer Group " Codon Preference ") of available commercially available acquisition to carry out statistical study.Referring to Devereaux, et al., (1984) Nucleic Acids Res.12:387-395 (people such as Devereaux,, " nucleic acids research ", the 12nd volume, 387-395 page or leaf in 1984); Or MacVector 4.1 (Eastman Kodak of New Haven, the Connecticut State (Eastman Kodak Co., New Haven, Conn.)).Thereby, the invention provides at least one the codon usage frequency characteristic of coding region in the polynucleotide of the present invention.Any integer from the number of 3 to this paper polynucleotide of the present invention that provide can be provided the number (3 Nucleotide of each amino acid) that can be used for determining the polynucleotide of codon usage frequency.Randomly, polynucleotide will be full length sequence.The exemplary number that is used for the sequence of statistical study can be at least 1,5,10,20,50 or 100.
Sequence reorganization
The invention provides the method for using polynucleotide of the present invention to carry out sequence reorganization and the composition that obtains thus.Sequence reorganization is announced among the No.96/19256 in the PCT patent description.Also can be referring to Zhang, et al., (1997) Proc.Natl.Acad.Sci.USA 94:4504-9 (people such as Zhang, 1997, " institute of NAS periodical ", the 94th volume, 4504-4509 page or leaf) and Zhao, et al., (1998) Nature Biotech 16:258-61 (people such as Zhao,, " Nature Biotechnol " in 1998, the 16th volume, the 258-261 page or leaf).In general, sequence reorganization provides the means for generation of the library of the polynucleotide with desired characteristic, can select or screens this library.Comprise the library that the correlated series polynucleotide of the sequence area that has sequence identity basically and can carry out homologous recombination in external or body produce recombination of polynucleotide from a group.The colony of the polynucleotide of sequence restructuring comprises the subgroup of the polynucleotide that have required or favourable characteristic and can select by suitable selection or screening method.Described characteristic can be any character or the attribute that can select or detect with screening system, can comprise following character: coded protein, transcribe element, control sequence, RNA processing, rna stability, chromatin conformation, gene or the genetically modified translation of transcribing or other express the character of character, reproduction element, protein bound element etc., for example give and to select or any feature of detectability matter.In certain embodiments, altered K for the wild-type protein that provides with respect to this paper will be provided the characteristic of selection mAnd/or K CatIn other embodiments, the ligand binding affinity that has of the protein that produces of sequence reorganization or polynucleotide will be than the height of the wild-type polynucleotide of non-reorganization.In other embodiment, to compare with the wild-type polynucleotide of non-reorganization, protein or polynucleotide that sequence reorganization produces will have altered best pH.The raising of this class character can account for wild offset at least 110%, 120%, 130%, 140% or be higher than 150%.
Recombinant expression cassettes
The present invention also provides the recombinant expression cassettes that comprises nucleic acid of the present invention.Can be with the nucleotide sequence of the required polynucleotide of the present invention of coding, for example code length be enough to encode cDNA or the genome sequence of polypeptide of active protein of the present invention is used for making up recombinant expression cassettes, this expression cassette can be introduced required host cell.Recombinant expression cassettes will comprise the polynucleotide of the present invention that effectively are connected to the transcription initiation regulating and controlling sequence usually, and described transcription initiation regulating and controlling sequence will guide described polynucleotide transcribing in the host cell (such as the tissue of conversion of plant) of expection.
For example, plant expression vector can comprise: (1) 5 ' and 3 ' regulating and controlling sequence transcribe plant gene and (2) the dominant selected marker thing of having cloned under the control.If necessary, this plant expression vector also (for example can contain the promoter regulation district, give inducible expression or constitutive expression, by environment or expression that grow to regulate, or cell or tissue specificity/selective expression's promoter regulation district), transcription initiation site, ribosome bind site, RNA processing signal, Transcription Termination site and/or polyadenylation signal.
Can adopt can the guiding polynucleotide of the present invention aftergrowth the institute in a organized way in the expression the plant promoter fragment.This promotor is referred to herein as " composing type " promotor and enlivens under most of envrionment conditionss and growth or cytodifferentiation state.The example of constitutive promoter comprises 1 of the T-DNA that comes from agrobacterium tumefaciens (Agrobacterium tumefaciens) ' or 2 ' promotor, Smas promotor, cinnamyl-alcohol dehydrogenase promotor (U.S. Patent No. 5,683,439), Nos promotor, rubisco promotor, GRP1-8 promotor, from the 35S promoter of cauliflower mosaic virus (CaMV), such as Odell, et al., (1985) the Nature 313:810-2 (people such as Odell, 1985, " nature ", the 313rd volume, the 810-812 page or leaf) described in; Rice actin (McElroy, et al., (1990) Plant Cell 163-171 (people such as McElroy, nineteen ninety, " vegetable cell ", 163-171 page or leaf)); Ubiquitin (Christensen, et al., (1992) Plant Mol.Biol.12:619-632 (people such as Christensen, 1992, " molecular biology of plants ", the 12nd volume, 619-632 page or leaf) and Christensen, et al., (1992) Plant Mol.Biol.18:675-89 (people such as Christensen,, " molecular biology of plants " in 1992, the 18th volume, the 675-689 page or leaf)); PEMU (Last, et al., (1991) Theor.Appl.Genet.81:581-8 (people such as Last,, " theory and applied genetics ", the 81st volume, 581-588 page or leaf in 1991)); MAS (Velten, et al., (1984) J.3:2723-30 (people such as Velten,, " European Molecular Bioglogy Organization's proceedings " in 1984 of EMBO, the 3rd volume, and corn H3 histone (Lepetit, et al., (1992) Mol.Gen.Genet.231:276-85 (people such as Lepetit the 2723-2730 page or leaf)), 1992, " MGG ", the 231st volume, 276-285 page or leaf) and Atanassvoa, et al., (1992) Plant Journal 2 (3): 291-300 (people such as Atanassvoa,, " plant magazine " in 1992, the 3rd phase of the 2nd volume, the 291-300 page or leaf)); The ALS promotor is as described in PCT public announcement of a patent application No.WO 96/30530; GOS2 (U.S. Patent No. 6,504,083) and other transcription initiation regions from various plants gene well known by persons skilled in the art.For the present invention, ubiquitin promoter is the preferred promoter of expressing for monocotyledons.
Perhaps, plant promoter can instruct expression or in addition the expression under more accurate environment or growth control of polynucleotide of the present invention in particular organization.This promotor is referred to herein as " induction type " promotor (Rab17, RAD29).Can realize comprising by the envrionment conditions of transcribing that inducible promoter carries out the existence of pathogenic agent attack, oxygen free condition or light.The example of inducible promoter is Adh1 promotor (its can by hypoxemia or cold stress-inducing), Hsp70 promotor (it can be induced by heat stress) and PPDK promotor (its can by photoinduction).
Only comprise or preferential initial promotor of transcribing in some tissue (such as leaf, root, fruit, seed or flower) at the example of growing the promotor under the control.Depend on promotor in genomic position, the operation of promotor also can change.Thereby inducible promoter is composing type wholly or in part at some position changeable.
If expression of polypeptides is required, then usually be desirably in 3 of polynucleotide encoding district '-end comprises the polyadenylation district.This polyadenylation district can come from the various plants gene, or comes from T-DNA.3 ' terminal sequence to be added can come from (for example) nopaline synthase or octopine synthase gene, perhaps comes from another plant gene, or more not preferably, is derived from any other eukaryotic gene.The example of these controlling elements includes but not limited to that 3 ' stops and/or poly-adenosine zone, agrobacterium tumefaciens nopaline synthase (no) gene (Bevan for example, et al., (1983) Nucleic Acids Res.12:369-85 (people such as Bevan, nineteen eighty-three, " nucleic acids research ", the 12nd volume, the 369-385 page or leaf)), potato proteinase inhibitor II (PINII) gene (Keil, et al., (1986) Nucleic Acids Res.14:5641-50 (people such as Keil, 1986, " nucleic acids research ", the 14th volume, 5641-5650 page or leaf) and An, et al., (1989) Plant Cell 1:115-22 (people such as An,, " vegetable cell " in 1989, the 1st volume, and CaMV 19S gene (Mogen, et al., (1990) Plant Cell 2:1261-72 (people such as Mogen the 115-122 page or leaf)), nineteen ninety, " vegetable cell ", the 2nd volume, 1261-1272 page or leaf)).
Intron sequences can be added into 5 ' non-translational region of part encoding sequence or encoding sequence to be increased in the amount of the ripe information of gathering in the cytosol.But the intron that comprises montage in the transcription unit in the animal and plant expression construct, having confirmed can both increase genetic expression and be up to 1000 times of (Buchman and Berg on the mRNA level and on the protein level, (1988) Mol.Cell Biol.8:4395-4405 (Buchman and Berg, 1988, " molecular cytobiology ", the 8th volume, the 4395-4405 page or leaf); Callis, et al., (1987) Genes Dev.1:1183-200 (people such as Callis,, " gene and growth ", the 1st volume, 1183-1200 page or leaf in 1987)).When being arranged near 5 of transcription unit ' end, the intron of this genetic expression strengthens normally maximum.The use of corn intron A dh1-S introne 1, Adh1-S intron 2 and Adh1-S intron 6, Bronze-1 intron is known in the art.Generally referring to THE MAIZE HANDBOOK, Chapter 116, Freeling and Walbot, eds., Springer, New York (1994) (" corn handbook, the 116th chapter, Freeling and Walbot (editor), Springer Verlag is published, New York, 1994).
The plant signal sequence includes but not limited to: coding is with the DNA/RNA sequence (Dratewka-Kos of the signal peptide of the extracellular matrix of protein targeted plants cell, et al., (1989) J.Biol.Chem.264:4896-900 (people such as Dratewka-Kos, 1989, " journal of biological chemistry ", the 264th volume, the 4896-4900 page or leaf)), for example wrinkle leaf tobacco (Nicotiana plumbaginifolia) extension gene (DeLoose, et al., (1991) Gene 99:95-100 (people such as DeLoose, 1991, " gene ", the 99th volume, 95-100 page or leaf)); With the signal peptide of protein target vacuole, sweet potato sporamin gene (Matsuka, et al. for example, (1991) Proc.Natl.Acad.Sci.USA 88:834 (people such as Matsuka,, " institute of NAS periodical " in 1991, the 88th volume, the 834th page)) and barley lectin plain gene (Wilkins, et al., (1990) Plant Cell, 2:301-13 (people such as Wilkins, nineteen ninety, " vegetable cell ", the 2nd volume, the 301-313 page or leaf)); Cause the secreted signal peptide of protein, PRIb signal peptide (Lind for example, et al., (1992) the Plant Mol.Biol.18:47-53 (people such as Lind, 1992, " molecular biology of plants ", the 18th volume, or barley α-amylase (BAA) (Rahmatullah, et al., (1989) Plant Mol.Biol.12:119 (people such as Rahmatullah the 47-53 page or leaf)), 1989, " molecular biology of plants ", the 12nd volume, the 119th page), be incorporated herein by reference) or with the signal peptide of protein target plastid, rape alkene acyl ACP reductase enzyme (Verwaert, et al., (1994) Plant Mol.Biol.26:189-202 (people such as Verwaert for example, 1994, " molecular biology of plants ", the 26th volume, 189-202 page or leaf)) can be used among the present invention.Merging extremely round the clock, the barley α-amylase signal sequence of polynucleotide is preferred constructs for expressing at corn of the present invention.
The carrier that comprises from the sequence of polynucleotide of the present invention will comprise marker gene usually, this marker gene can be on vegetable cell that imparts selective phenotype.Usually, selected marker's antibiotics resistance of will encoding, suitable gene comprises that coding is to the gene (for example aada gene) of this antibiotic resistance of spectinomycin, streptomycin phosphotransferase (SPT) gene of coding streptomycin resistance, neomycin phosphotransferase (NPTII) gene of coding kantlex or Geneticin resistance, hygromix phosphotransferase (HPT) gene of coding hygromycin resistance, coding is to playing the weedicide of the effect that suppresses acetolactate synthase (ALS), the gene of the resistance of sulfonylurea herbicide (for example containing sudden change acetolactate synthase (ALS) gene of S4 and/or Hra sudden change particularly that causes this resistance) particularly, coding is to the gene (for example bar gene) of the resistance of the weedicide that plays the effect that suppresses glutamine synthase such as glufosinates or basta, or other this genes known in the art.The bar genes encoding is to the resistance of weedicide basta, and the als gene coding is to the resistance of chlorsulfuron.
Be used in that the typical carriers of expressing gene is well known in the art in the higher plant, comprise the carrier that spreads out from tumor inducing (Ti) plasmid of agrobacterium tumefaciens, such as Rogers, et al., (1987) Meth.Enzymol.153:253-77 (people such as Rogers,, " Enzymology method " in 1987, the 153rd volume, 253-277 page or leaf) describe.These carriers are plant integration type carriers, because when transforming, these carriers are integrated into the part of carrier DNA in the genome of host plant.Can be used for exemplary agrobacterium tumefaciens carrier of the present invention is Schardl, et al., (1987) Gene 61:1-11 (people such as Schardl, 1987, " gene ", the 61st volume, the 1-11 page or leaf) and Berger, et al., (1989) Proc.Natl.Acad.Sci.USA, the 86:8402-6 (people such as Berger, 1989, " institute of NAS periodical ", the 86th volume, 8402-8406 page or leaf) described plasmid pKYLX6 and pKYLX7.Available another kind of carrier is plasmid pBI101.2 among the present invention, and it can derive from the CLONTECH Laboratories of California Paro Austria many (Palo Alto, CA), Inc..
The expression of protein in host cell
Use nucleic acid of the present invention, can be at cell such as bacterial cell, yeast cell, insect cell, mammalian cell or the preferred plant cells protein of the present invention of recombined engineering transformation.This class cell (for example, aspect quantity, composition, position and/or time) under the non-natural condition produces protein, because they are become to produce protein under the non-natural condition by hereditary change by human intervention.
Can expect that those skilled in the art knows the multiple expression system that can be used for expressing the nucleic acid of coding protein of the present invention.Have no intention to describe in detail the whole bag of tricks that becomes known for marking protein in prokaryotic organism or eukaryote.
Simplified summary, the expression of the isolating nucleic acid of code book invention protein usually can, effectively be connected to promotor (composing type or induction type), then be integrated in the expression vector and realize by for example making DNA or cDNA.This carrier can be suitable for copying and integrating in prokaryotic organism or eukaryote.Typical expression vector contains transcribing and translation termination, homing sequence and promotor of the expression that can be used for regulating and control code book invention protein DNA.In order to obtain the high level expression of clone gene, expectation construction of expression vector, this expression vector contain the strong promoter (such as ubiquitin promoter) of transcribing in order to guidance at minimum level, be used for the ribosome bind site of translation initiation and transcribe/translation termination.Constitutive promoter is classified as can provide a series of constitutive expressions.Thereby some is weak constitutive promoter, and other are strong constitutive promoters.In general, so-called " weak promoter " means to drive encoding sequence with the promotor of low expression level.So-called " low-level " means to be in the level of about 1/10,000 transcript to about 1/100,000 transcript to about 1/500,000 transcript.On the contrary, " strong promoter " driving encoding sequence is expressed with " high level " or about 1/10 transcript to about 1/100 transcript to about 1/1,000 transcript.
The technician will recognize, can modify protein of the present invention and not lower its biological activity.Can carry out some clone who modifies to be conducive to target molecule, express or mix in fusion rotein.This modification is well-known to those skilled in the art, for example comprise at N-terminal and add methionine(Met) so that initiation site to be provided, or extra amino acid (for example poly His) is set to produce expediently restriction site or terminator codon or the purifying sequence of location at arbitrary end.
Expression in prokaryotic organism
Prokaryotic cell prokaryocyte can be used as the host of expression.Prokaryotic organism are the most normal by multiple coli strain representative; Yet, also can use other microorganism strains.Be defined as in this article the protokaryon control sequence commonly used of the promotor that comprises for transcription initiation (optional have operon) and ribosome bind site sequence, comprise such as β-lactamase (penicillinase) promoter systems and lactose (lac) promoter systems (Chang, et al., (1977) the Nature 198:1056 (people such as Chang, 1977, " nature ", the 198th volume, the 1056th page)), tryptophane (trp) promoter systems (Goeddel, et al., (1980) Nucleic Acids Res.8:4057 (people such as Goeddel,, " nucleic acids research " in 1980, the 8th volume, the 4057th page)) and λ derive promotor commonly used and N-gene ribosome bind site (Shimatake, et al., (1981) Nature 292:128 (people such as Shimatake of PL promotor and so on, 1981, " nature ", the 292nd volume, the 128th page)).It also is useful comprising the selection marker thing in dna vector in the intestinal bacteria is advanced in transfection.The example of this mark comprises that regulation is to the gene of the resistance of penbritin, tsiklomitsin or paraxin.
Select carrier so that the concern gene is incorporated in the suitable host cell.Bacteria carrier is plasmid or phage origin normally.With suitable bacterial cell with phage vector particle transfection or with naked phage vector DNA transfection.If the use plasmid vector is then used bacterial cell plasmid vector DNA transfection.Be used for expressing protein expression of the present invention system and can use bacillus (Bacillus sp.) and salmonella (Salmonella) (Palva, et al., (1983) the Gene 22:229-35 (people such as Palva, nineteen eighty-three, " gene ", the 22nd volume, the 229-235 page or leaf); Mosbach, et al., (1983) Nature 302:543-5 (people such as Mosbach, nineteen eighty-three, " nature ", the 302nd volume, 543-545 page or leaf)).The pGEX-4T-1 plasmid vector that derives from Pharmacia (Pharmacia) is preferred coli expression carrier of the present invention.
Expression in eukaryote
Multiple eukaryotic expression system such as yeast, insect cell line, plant and mammalian cell are well known by persons skilled in the art.Such as following simplicity of explanation, the present invention can express in these eukaryotic systems.In certain embodiments, with transform/vegetable cell (as hereinafter discussing) of transfection is as expression system, for generation of protein of the present invention.
Synthetic in yeast of heterologous protein is well-known.Sherman, et al., (1982) METHODS IN YEAST GENETICS, the Cold Spring Harbor Laboratory (people such as Sherman, nineteen eighty-two, " yeast genetics method ", cold spring harbor laboratory) be to describe the multiple works that produces the extensive approval of method of protein in the yeast that is used in.Two kinds of yeast for generation of eukaryotic protein that extensively adopt are yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) and pichia pastoris phaff (Pichia pastoris).Be used for being known in the art and can obtaining from commercial source (for example hero company (Invitrogen)) in carrier, bacterial strain and the method for yeast belong (Saccharomyces) and Pichia (pichia) expression.As required, suitable carrier has expression control sequenc usually, for example promotor (comprising glycerol 3-phosphate acid kinase or alcohol oxidase promotor) and ori, terminator sequence etc.
Protein of the present invention in case express, can come from yeast separation by lysing cell and to lysate or centrifugal sediment application standard protein stripping technique.Can be by finish the monitoring to purge process with the radioimmunoassay of protein imprinted technology or other standards immunoassay.
Also the sequence of coding protein of the present invention can be connected to multiple for the transfection expression vector of the cell culture of Mammals, insect or plant origin for example.Mammalian cell system can be the form of monolayer cell usually, but also can use mammalian cell suspension.This area has been developed multiple suitable host clone that can the expressed protein, comprises HEK293, BHK21 and Chinese hamster ovary celI system.The expression vector that is used for these cells can comprise expression control sequenc, such as replication orgin, promotor (CMV promotor for example, HSV tk promotor or pgk (phosphoglyceric kinase) promotor), enhanser (Queen, et al., (1986) Immunol.Rev.89:49 (people such as Queen, 1986, " immunology comment ", the 89th volume, the 49th page)) and necessary machining information site such as ribosome bind site, the RNA splice site, polyadenylation site (for example the large T Ag of SV40 poly A adds the site) and Transcription Termination subsequence.Other zooblasts that can be used for producing protein of the present invention can obtain from for example American type culture collection (American Type Culture Collection) Catalogue of Cell Lines and Hybridomas (" clone and hybridoma catalogue ") (the 7th edition, 1992).
Be used for usually coming from the SF9 baculovirus at the suitable carrier of expressed in insect cells protein of the present invention.Suitable insect cell line comprises that mosquito larvae, silkworm, armyworm, moth and Drosophila (Drosophila) clone such as Schneider clone are (referring to for example Schneider, (1987) J.Embryol.Exp.Morphol.27:353-65 (Schneider, 1987, " fetology and experimental morphology magazine ", the 27th volume, the 353-365 page or leaf)).
As use the yeast, when adopting higher animal or plant host cell, usually polyadenylation or Transcription Termination subsequence are integrated in the carrier.The example of terminator sequence is the Polyadenylation sequence from bovine growth hormone gene.Also can comprise the sequence for the accurate montage of transcript.An example of montage sequence is the VP1 intron (Sprague, et al., (1983) J.Virol.45:773-81 (people such as Sprague, nineteen eighty-three, " Journal of Virology ", the 45th volume, 773-781 page or leaf)) from SV40.In addition, the gene order that copies that is controlled in the host cell can be integrated into carrier, those (Saveria-Campo that exist in the carrier such as bovine papillomavirus type, " Bovine Papilloma Virus DNA a Eukaryotic Cloning Vector " (bovine papilloma virus DNA: a kind of eukaryotic cloning carrier), be stated from DNA CLONING:A PRACTICAL APPROACH, vol.II, Glover, ed., IRL Press, Arlington, VA, (Glover edits pp.213-38 (1985) for " dna clone: a kind of practical approach ", II volume, IRL press, the Arlington, Virginia, 213-238 page or leaf, 1985)).
In addition, the Circadian Expression gene that is arranged in the suitable plant expression vector can be used for transformed plant cells.Then can maybe the cell that transforms can be used for the regeneration of transgenic plant from the plant callus isolated polypeptide.Can gather in the crops this transgenic plant, suitable tissue (for example, seed or leaf) be carried out large-scale protein matter extract and purification technique.
Methods for plant transformation
Multiple method for alien gene being changed over to plant is arranged is known and can be used to round the clock multinuclear glycosides 1 acid is inserted in the plant host, and this comprises biology and physical Plant Transformation scheme.Referring to such as people such as Miki, " Procedure for Introducing Foreign DNA into Plants " (method of foreign DNA introduced plant), be stated from METHODS IN PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY, Glick and Thompson, eds., CRC Press, Inc., Boca Raton, and pp.67-88 (1993) (" molecular biology of plants and biotechnological means ", Glick and Thompson edit, CRC press, Boca Raton, 67-88 page or leaf, 1993).Selected method changes with host plant, the transgenosis, the transgenosis of microbe-mediated such as the agriculture bacillus mediated transgenosis (Horsch that comprise the mediation of chemical transfection method such as calcium phosphate, et al., Science 227:1229-31 (the 1985) (people such as Horsch, " science ", the 227th volume, 1229-1231 page or leaf, 1985)), electroporation, microinjection and particle gun bombardment.
Being used for the expression cassette of vegetable cell or metaplasia and plant regeneration and carrier and extracorporeal culturing method is known and can obtains.Referring to such as people such as Gruber, " Vectors for Plant Transformation " (carrier that is used for Plant Transformation), be stated from METHODS IN PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY (" molecular biology of plants and biotechnological means ") (ibid), the 89-119 page or leaf.
Can be generally used for directly sending in the polynucleotide or polypeptide introduced plant of technology with separation of cell by one or more.The type (being monocotyledons or dicotyledons) that depends on organism, cell, plant or the vegetable cell that will carry out genetic modification, this mode can be different.The appropriate method of transformed plant cells comprises microinjection (Crossway, et al., (1986) the Biotechniques 4:320-334 (people such as Crossway, 1986, " biotechnology ", the 4th volume, the 320-334 page or leaf) and U.S. Patent No. 6,300,543), electroporation (Riggs, et al., (1986) Proc.Natl.Acad.Sci.USA 83:5602-5606 (people such as Riggs, 1986, " institute of NAS periodical ", the 83rd volume, the 5602-5606 page or leaf)), direct gene transfer (Paszkowski, et al., (1984) EMBO (people such as Paszkowski J.3:2717-2722,1984, " European Molecular Bioglogy Organization's proceedings ", the 3rd volume, the 2717-2722 page or leaf)) and the trajectory particle accelerate (referring to such as people such as Sanford, U.S. Patent No. 4,945,050; WO 91/10725 and McCabe, et al., (1988) Biotechnology6:923-926 (people such as McCabe,, " biotechnology ", the 6th volume, 923-926 page or leaf in 1988)).Also can be referring to people such as Tomes, Direct DNA Transfer into Intact Plant Cells Via Microprojectile Bombardment (directly DNA being transferred in the intact plant by microparticle bombardment), be stated from Plant Cell, Tissue and Organ Culture, Fundamental Methods eds.Gamborg and Phillips, Springer-Verlag Berlin Heidelberg New York, 1995 (" vegetable cells, tissue and organ culture: basic skills ", Gamborg and Phillips edit, Springer Verlag press Berlin Heidelberg, New York, nineteen ninety-five) the 197-213 page or leaf; U.S. Patent No. 5,736,369 (meristematic tissue); Weissinger, et al., (1988) Ann.Rev.Genet.22:421-477 (people such as Weissinger,, " heredity is commented academic year ", the 22nd volume, 421-477 page or leaf in 1988); Sanford, et al., (1987) Particulate Science and Technology 5:27-37 (people such as Sanford,, " particle science and technology ", the 5th volume, 27-37 page or leaf in 1987) (onion); Christou, et al., (1988) Plant Physiol.87:671-674 (people such as Christou,, " plant physiology ", the 87th volume, 671-674 page or leaf in 1988) (soybean); Datta, et al., (1990) Biotechnology 8:736-740 (people such as Datta, nineteen ninety, " biotechnology ", the 8th volume, 736-740 page or leaf) (paddy rice); Klein, et al., (1988) Proc.Natl.Acad.Sci.USA 85:4305-4309 (people such as Klein,, " institute of NAS periodical ", the 85th volume, 4305-4309 page or leaf in 1988) (corn); Klein, et al., (1988) Biotechnology 6:559-563 (people such as Klein,, " biotechnology ", the 6th volume, 559-563 page or leaf in 1988) (corn); WO 91/10725 (corn); Klein, et al., (1988) Plant Physiol.91:440-444 (people such as Klein,, " plant physiology ", the 91st volume, 440-444 page or leaf in 1988) (corn); Fromm, et al., (1990) Biotechnology 8:833-839 (people such as Fromm, nineteen ninety, " biotechnology ", the 8th volume, 833-839 page or leaf); And Gordon-Kamm, et al., (1990) Plant Cell 2:603-618 (people such as Gordon-Kamm, nineteen ninety, " vegetable cell ", the 2nd volume, 603-618 page or leaf) (corn); Hooydaas-Van Slogteren and Hooykaas (1984) Nature (London) 311:763-764 (Hooydaas-Van Slogteren and Hooykaas,, " nature ", London, the 311st volume, 763-764 page or leaf in 1984); Bytebier, et al., (1987) Proc.Natl.Acad.Sci.USA 84:5345-5349 (people such as Bytebier,, " institute of NAS periodical ", the 84th volume, 5345-5349 page or leaf in 1987) (Liliaceae); De Wet, et al., (1985) In The Experimental Manipulation of Ovule Tissues, ed.Chapman, et al., pp.197-209; Longman, and NY (people such as De Wet, 1985, " experimental implementation of ovule tissue ", the people such as Chapman edit, 197-209 page or leaf, Longman, New York) (pollen); Kaeppler, et al., (1990) Plant Cell Reports 9:415-418 (people such as Kaeppler, nineteen ninety, " vegetable cell report ", the 9th volume, 415-418 page or leaf); And Kaeppler, et al., (1992) Theor.Appl.Genet.84:560-566 (people such as Kaeppler,, " theory and applied genetics ", the 84th volume, 560-566 page or leaf in 1992) (conversion of whisker mediation); U.S. Patent No. 5,693,512 (ultrasonic degradations); D ' Halluin, et al., (1992) Plant Cell 4:1495-1505 (D ' people such as Halluin, 1992, " vegetable cell ", the 4th volume, 1495-1505 page or leaf) (electroporation); Li, et al., (1995) Plant Cell Reports 12:250-255 (people such as Li, nineteen ninety-five, " vegetable cell report ", the 12nd volume, 250-255 page or leaf); And Christou and Ford, (1993) Annals of Botany 75:407-413 (Christou and Ford,, " phytology yearbook ", the 75th volume, 407-413 page or leaf in 1993) (paddy rice); Osjoda, et al., (1996) Nature Biotech.14:745-750 (people such as Osjoda,, " Nature Biotechnol ", the 14th volume, 745-750 page or leaf in 1996); Agriculture bacillus mediated corn transforms (U.S. Patent No. 5,981,840); Silicon carbide whisker method (Frame, et al., (1994) Plant be (people such as Frame,, " plant magazine ", the 6th volume, 941-948 page or leaf in 1994) J.6:941-948); Laser means (Guo, et al., (1995) Physiologia Plantarum 93:19-24 (people such as Guo, nineteen ninety-five, " plant physiology ", the 93rd volume, 19-24 page or leaf)); Ultrasonic degradation method (Bao, et al., (1997) Ultrasound in Medicine ﹠amp; Biology 23:953-959 (people such as Bao,, " Med Biol is ultrasonic ", the 23rd volume, 953-959 page or leaf in 1997); Finer and Finer, (2000) Lett Appl Microbiol.30:406-10 (Finer and Finer,, " applied microbiology wall bulletin ", the 30th volume, 406-410 page or leaf in 2000); Amoah, et al., (2001) J Exp Bot 52:1135-42) (people such as Amoah, calendar year 2001, " experimental botany magazine ", the 52nd volume, 1135-1142 page or leaf); Polyoxyethylene glycol method (Krens, et al., (1982) Nature 296:72-77 (people such as Krens, nineteen eighty-two, " nature ", the 296th volume, 72-77 page or leaf)); The protoplasma of unifacial leaf and dicotyledons cell can be used electroporation (Fromm, et al., (1985) the Proc.Natl.Acad.Sci.USA 82:5824-5828 (people such as Fromm, 1985, " institute of NAS periodical ", the 82nd volume, and microinjection (Crossway the 5824-5828 page or leaf)), et al., (1986) Mol.Gen.Genet.202:179-185 (people such as Crossway, 1986, " MGG ", the 202nd volume, the 179-185 page or leaf)) transform, these documents are all incorporated this paper by reference into.
Agriculture bacillus mediated conversion
The most widely used natural conversion system that method in the expression vector introduced plant is based on Agrobacterium.Agrobacterium tumefaciens and Agrobacterium rhizogenes are the plant-pathogenic soil bacterias, and it can the genetic transformation plant cell.Agrobacterium tumefaciens and Agrobacterium rhizogenes Ti and the Ri plasmid gene that carries the genetic transformation of being responsible for plant separately.Referring to for example Kado, (1991) Crit.Rev.Plant Sci.10:1 (Kado,, " plant science comment ", the 10th volume, page 1 in 1991).The agrobacterium vector system and method for relevant agriculture bacillus mediated transgenosis be described in to provide in the Publication about Document: the people such as Gruber (ibid); The people such as Miki (ibid); And Moloney, et al., (1989) Plant Cell Reports8:238 (people such as Moloney,, " vegetable cell report ", the 8th volume, the 238th page in 1989).
Similarly, the gene insertion can be come from agrobacterium tumefaciens or Agrobacterium rhizogenes Ti separately or the T-DNA district of Ri plasmid.Thereby, can use these plasmids, as above the construction expression box.Known have many control sequences, and it is when being coupled to allogeneic coding sequence and transform in the host organisms, at the fidelity that demonstrates genetic expression aspect the tissue/organ specificity of initial code sequence.Referring to for example Benfey and Chua, (1989) Science 244:174-81 (Benfey and Chua,, " science ", the 244th volume, 174-181 page or leaf in 1989).The specially suitable control sequence that is used for these plasmids is in the specific expressed promotor of the composing type leaf of various target plants for gene.Other available control sequences comprise promotor and the terminator from nopaline synthase gene (NOS).NOS promotor and terminator are present among the plasmid pARC2, and this plasmid can derive from American type culture collection, and the ATCC number of depositing of appointment is 67238.If use a kind of like this system, then also must exist from virulence (vir) gene of Ti or Ri plasmid, perhaps with the T-DNA part, perhaps be present in double element system on the independent carrier by vir gene wherein.This type systematic, wherein the method for used carrier and transformed plant cells is in U.S. Patent No. 4,658,082; The U.S. Patent application No.913 that on October 1st, 1986 submitted to, 914, they are in the U.S. Patent No. 5,262 that is published on November 16th, 1993,306 and Simpson, et al., (1986) Plant Mol.Biol.6:403-15 (people such as Simpson, 1986, " molecular biology of plants ", the 6th volume, 403-415 page or leaf) describe to some extent in (also quoting in ' 306 patents), described document all is incorporated herein by reference in full.
In case make up, these plasmids can be placed Agrobacterium rhizogenes or agrobacterium tumefaciens and these carriers are used for the cell of conversion of plant species, it is susceptible that the cell of described plant species infects fusarium (Fusarium) or Alternaria (Alternaria) usually.The present invention it will also be appreciated that some other transgenic plant, includes but not limited to soybean, corn, Chinese sorghum, clover, paddy rice, trifolium, Caulis et Folium Brassicae capitatae, banana, coffee, celery, tobacco, cowpea, cotton, muskmelon and pepper.The selection of agrobacterium tumefaciens or Agrobacterium rhizogenes will be depended on the plant with its conversion.Usually, agrobacterium tumefaciens is for the preferred organism that transforms.It is susceptible that much several dicotyledonss, some gymnosperms and minority monocotyledons (for example some member of Liliales (Liliales) and Arales (Arales)) infect agrobacterium tumefaciens.Agrobacterium rhizogenes also has widely host range, contains most of dicotyledonss and some gymnosperms, and it comprises the member of pulse family (Leguminosae), composite family (Compositae) and Chenopodiaceae (Chenopodiaceae).Monocotyledons can certain success ratio transform now.European patent application No.604662A1 discloses with the monocotyledonous method of Agrobacterium-mediated Transformation.European patent application No.672 752A1 discloses the scultellum monocotyledonous method of Agrobacterium-mediated Transformation of using immature embryo.The people such as Ishida have discussed method (the Nature Biotechnology 14:745-50 (1996) (" Nature Biotechnol " that comes maize transformation by making immature embryo be exposed to agrobacterium tumefaciens, the 14th volume, the 745-750 page or leaf, 1996)).
In case transform, these cells can be used for the regeneration of transgenic plant.For example, whole plant can produce wound by making this plant, then carrier is introduced this wound site and is come to infect with these carriers.Can make any part of plant produce wound, comprise leaf, stem and root.Perhaps, can be with the plant tissue of explant form such as cotyledon tissue or leaf disk with these carriers inoculations, and under the condition that can promote plant regeneration, cultivate.Can be with being used as plant origin by root or the seedling that transforms with Agrobacterium rhizogenes or agrobacterium tumefaciens (gene that contains coding fumonisin degrading enzyme) inoculation plant tissue, with the fumonisin resistant transgenic plants of regenerating by somatic embryo Somatic Embryogenesis or organ.The example of these class methods of aftergrowth tissue is disclosed in Shahin, Theor.Appl.Genet.69:235-40 (1985) (Shahin, " theory and applied genetics ", the 69th volume, 235-240 page or leaf); U.S. Patent No. 4,658,082; The people such as Simpson (the same) and the U.S. Patent application No.913 that all submits on October 1st, 1986,913 and 913,914, as be published in the U.S. Patent No. 5 on November 16th, 1993,262,306 quote, and incorporate by reference whole disclosures of above-mentioned document into this paper.
Direct gene transfer
Although the host range of agriculture bacillus mediated conversion is extensive, but the cereal crop species that some are main and gymnosperm are stupid stubborn for this transgenosis pattern generally, although in rice, obtained recently certain success (Hiei, et al., (1994) The Plant Journal 6:271-82 (people such as Hiei,, " plant magazine " in 1994, the 6th volume, the 271-282 page or leaf)).Developed method (being referred to as direct gene transfer) conduct the substituting agriculture bacillus mediated conversion that several plant transforms.
General applicable methods for plant transformation is the conversion of little projectile body (microprojectile) mediation, and wherein DNA is carried on the surface of little projectile body of about 1 to 4 μ m.With particle gun device (biolistic device) with in the expression vector introduced plant tissue, this particle gun device accelerates to little projectile body the speed of 300-600m/s, this speed is enough to penetrate plant cell wall and film (Sanford, et al., (1987) Part.Sci.Technol.5:27 (people such as Sanford,, " particle science and technology " in 1987, the 5th volume, the 27th page); Sanford, (1988) Trends Biotech 6:299 (Sanford,, " biotechnology trend ", the 6th volume, the 299th page in 1988); Sanford, (1990) Physiol.Plant 79:206 (Sanford, nineteen ninety, " plant physiology ", the 79th volume, the 206th page) and Klein, et al., (1992) Biotechnology 10:268 (people such as Klein, 1992, " biotechnology ", the 10th volume, the 268th page)).
Physical delivery DNA is such as Zang to the another kind of method of plant, et al., the supersound process to target cell described in (1991) BioTechnology 9:996 (people such as Zang,, " biotechnology ", the 9th volume, the 996th page in 1991).Perhaps, the fusion of liposome or spheroplast is used for the expression vector introduced plant.Referring to for example Deshayes, et al., (1985) EMBO be the (people such as Deshayes J.4:2731,1985, " European Molecular Bioglogy Organization's proceedings ", the 4th volume, the 2731st page) and Christou, et al., (1987) Proc.Natl.Acad.Sci.USA 84:3962 (people such as Christou,, " institute of NAS periodical " in 1987, the 84th volume, the 3962nd page).Utilize CaCl 2Precipitation, polyvinyl alcohol or poly--L-Orn are directly taken in DNA in the protoplastis and are had been reported.Referring to for example Hain, et al., (1985) Mol.Gen.Genet.199:161 (people such as Hain, 1985, " MGG ", the 199th volume, the 161st page) and Draper, et al., (1982) Plant Cell Physiol.23:451 (people such as Draper, nineteen eighty-two, " plant cell physiology ", the 23rd volume, the 451st page).
The electroporation of protoplastis and intact cell and tissue is existing the description also.Referring to such as the people such as Donn (1990), be published in Abstracts of the VIIth Int ' l.Congress on Plant Cell and Tissue Culture IAPTC, A2-38, p.53 (" VII plant cell and tissue culture IAPTC conference summary ", the A2-38 volume, the 53rd page); D ' Halluin, et al., (1992) Plant Cell 4:1495-505 (D ' people such as Halluin, 1992, " vegetable cell ", the 4th volume, 1495-1505 page or leaf) and Spencer, et al., (1994) Plant Mol.Biol.24:51-61 (people such as Spencer,, " molecular biology of plants " in 1994, the 24th volume, the 51-61 page or leaf).
Increase round the clock activity and/or the level of the round the clock polypeptide of polynucleotide encoding
The activity of the round the clock polypeptide that increases round the clock polynucleotide encoding of the present invention and/or the method for level are provided.Can by will be round the clock polypeptide offer plant, realize level and/or the active increase of round the clock polypeptide of the present invention.This round the clock polypeptide can provide in the following way: will encode this round the clock the aminoacid sequence of polypeptide introduce in this plant, the nucleotide sequence of polypeptide of encoding is round the clock introduced in this plant, perhaps modify the genome seat of coding round the clock polypeptide of the present invention.
Discussed such as this paper other places, known in the art have several different methods to be used for polypeptide is offered plant, include but not limited to in the direct introduced plant of polypeptide coding to be had in the polynucleotide constructs introduced plant of polypeptide of cell number regulation activity (of short duration introducing or stable the introducing).Also recognize, method of the present invention can adopt can not be in the plant that transforms the polynucleotide of expression of pilot protein matter or RNA.Therefore, can encoding round the clock by change, gene or its promotor of polypeptide improve round the clock level and/or the activity of polypeptide.Referring to for example Kmiec, U.S. Patent No. 5,565,350; The people such as Zarling, PCT/US93/03868.Therefore, provide the plant of the mutagenesis of carrying the sudden change in the gene round the clock, wherein said sudden change increases the expression of gene round the clock or increases plant-growth and/or the allelotaxis of round the clock polypeptide of coding active.
Reduce round the clock activity and/or the level of polypeptide
Provide method to come to reduce or eliminate the activity of round the clock polypeptide of the present invention by can suppress the expression cassette transformed plant cells of the polynucleotide of the round the clock expression of polypeptide with expression.These polynucleotide can be by preventing that the translation of messenger RNA(mRNA) directly suppresses the round the clock expression of polypeptide round the clock, and perhaps suppressing to encode round the clock by coding, the polypeptide of transcribing or translating of the round the clock gene of polypeptide suppresses the round the clock expression of polypeptide indirectly.Being used for suppressing or eliminating gene is known in the art in the method for the expression of plant, and any this method can be used for suppressing among the present invention the round the clock expression of polypeptide.
According to the present invention, if round the clock the protein level of polypeptide for this with the diel polypeptide not through genetic modification or mutagenesis with the protein level in the plant that suppresses this round the clock expression of polypeptide less than 70%, then the expression of polypeptide is suppressed round the clock.In specific embodiments of the invention, this round the clock polypeptide according to the protein level in the modified plant of invention, for this with the diel polypeptide the plant that does not belong to mutant or through genetic modification with the protein level in the plant that suppresses this round the clock expression of polypeptide less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5% or less than 2%.The expression level of polypeptide can for example directly be measured by the level that detects the round the clock polypeptide of expressing in vegetable cell or the plant round the clock, perhaps for example pass through to measure plant-growth and/or allelotaxis's activity of the round the clock polypeptide in vegetable cell or the plant, or indirectly measure by the biomass of measuring in the plant.The method of carrying out this detection is described at the elsewhere of this paper.
In other embodiments of the invention, by reducing with the expression cassette transformed plant cells or eliminating the round the clock activity of polypeptide, described expression cassette comprises coding can suppress the round the clock polynucleotide of the polypeptide of polypeptide active.According to the present invention, if round the clock the plant-growth of polypeptide and/or allelotaxis active for this with the diel polypeptide not through modification with the plant-growth in the plant that suppresses this round the clock plant-growth of polypeptide and/or allelotaxis's activity and/or allelotaxis's activity less than 70%, the then round the clock plant-growth of polypeptide and/or allelotaxis's activity inhibited.In specific embodiments of the invention, plant-growth and/or the allelotaxis of polypeptide in modified plant according to the present invention is active round the clock for this, for this with the diel polypeptide not through modification with the plant-growth in the plant that suppresses this round the clock expression of polypeptide and/or allelotaxis's activity less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10% or less than 5%.When the plant-growth of polypeptide round the clock and/or allelotaxis's activity can not be measured by the described detection method of this paper elsewhere, then should activity according to the present invention by " elimination ".Determine that the round the clock plant-growth of polypeptide and/or the method for allelotaxis's activity are described at this paper elsewhere.
In other embodiments, can reduce or eliminate the round the clock activity of polypeptide by the destruction gene of polypeptide of encoding round the clock.The plant of the mutagenesis of carrying the sudden change in the gene is round the clock contained in the present invention, and wherein said sudden change reduces the expression of gene round the clock or suppress plant-growth and/or the allelotaxis of round the clock polypeptide of coding active.
Thereby, there are many methods to can be used for reducing or eliminate the round the clock activity of polypeptide.In addition, there is more than a kind of method to can be used for reducing and singly plants the round the clock activity of polypeptide.Reduce or eliminate round the clock that the non-limitative example of the method for the expression of polypeptide provides below.
1. based on the method for polynucleotide:
In some embodiments of the invention, use the expression cassette conversion of plant, this expression cassette can be expressed the polynucleotide of the expression that can suppress round the clock polypeptide of the present invention.Term used herein " expression " refers to the biosynthesizing of gene product, comprises transcribing and/or translating of described gene product.For example, for purposes of the present invention, can express the expression cassette of the polynucleotide of the expression that can suppress at least a round the clock polypeptide, be the expression cassette that can produce the RNA molecule of transcribing and/or translating that can suppress at least a round the clock polypeptide of the present invention.Protein or polypeptide refer to that from " expression " or " generation " of dna molecular this encoding sequence transcribes and translate and produce this protein or polypeptide, and protein or polypeptide refer to this RNA encoding sequence translation and produce protein or polypeptide from RNA molecule " expression " or " generation ".
Can suppressing round the clock, the example of the polynucleotide of the expression of polypeptide provides below.
I., justice inhibition/co-suppression is arranged
In some embodiments of the invention, the round the clock inhibition of expression of polypeptides can be by having justice to suppress or the co-suppression acquisition.For co-suppression, expression cassette is designed to express such RNA molecule, this RNA molecule is with all or part of corresponding to the messenger RNA(mRNA) of the polypeptide round the clock of encoding of " justice is arranged " orientation.The expression minimizing that can cause natural gene is expressed in crossing of this RNA molecule.Therefore, a plurality of plant that transform with this co-suppression expression cassette are screened to differentiate that those demonstrate the plant that the maximum of expression of polypeptides is round the clock suppressed.
The polynucleotide that are used for co-suppression can be corresponding to all or part of, 5 ' and/or the two all or part of of the encoding sequence of 3 ' non-translational region all or part of or the transcript of polypeptide round the clock of encoding and non-translational region of polypeptide transcript round the clock of the sequence of the polypeptide round the clock of encoding.These polynucleotide comprise among some all or part of embodiment of the coding region of polypeptide round the clock therein, expression cassette are designed to eliminate the initiator codon of these polynucleotide, so that can not translate protein.
Co-suppression can be used to suppress the expression of plant gene to produce for the plant that has undetectable protein level for the protein of these genes encodings.Referring to (for example) Broin, et al., (2002) Plant Cell 14:1417-1432 (people such as Broin,, " vegetable cell ", the 14th volume, 1417-1432 page or leaf in 2002).Co-suppression also can be used to suppress the expression of the multiple proteins in the same plant.Referring to (for example) U.S. Patent No. 5,942,657.The method that suppresses the expression of the native gene in the plant with co-suppression has description: Flavell in Publication about Document and patent, et al., (1994) the Proc.Natl.Acad.Sci.USA 91:3490-3496 (people such as Flavell, 1994, " institute of NAS periodical ", the 91st volume, the 3490-3496 page or leaf); Jorgensen, et al., (1996) Plant Mol.Biol.31:957-973 (people such as Jorgensen,, " molecular biology of plants ", the 31st volume, 957-973 page or leaf in 1996); Johansen and Carrington (2001) Plant Physiol.126:930-938 (Johansen and Carrington, calendar year 2001, " plant physiology ", the 126th volume, 930-938 page or leaf); Broin, et al., (2002) Plant Cell 14:1417-1432 (people such as Broin,, " vegetable cell ", the 14th volume, 1417-1432 page or leaf in 2002); Stoutjesdijk, et al., (2002) Plant Physiol.129:1723-1731 (people such as Stoutjesdijk,, " plant physiology ", the 129th volume, 1723-1731 page or leaf in 2002); Yu, et al., (2003) Phytochemistry63:753-763 (people such as Yu,, " vegetable chemistry " in 2003, the 63rd volume, the 753-763 page or leaf) and U.S. Patent No. 5,034,323,5,283,184 and 5,942,657, these documents and patent are incorporated herein by reference.The efficient of co-suppression can by in expression cassette have 3 of adopted sequence ' and 5 ' position of polyadenylation signal comprise that the poly-dT district improves.Referring to U.S. Patent Application Publication No.2002/0048814, be incorporated by reference this paper.Usually, the sequence of the transcript of this nucleotide sequence and native gene has sizable sequence identity, preferably be higher than about 65% sequence identity, more preferably be higher than about 85% sequence identity, most preferably be higher than about 95% sequence identity.Referring to U.S. Patent No. 5,283,184 and 5,034,323, incorporate by reference them into this paper.
Ii. Antisense Suppression
In some embodiments of the invention, can obtain by Antisense Suppression the round the clock inhibition of expression of polypeptides.For Antisense Suppression, expression cassette is designed to express and this round the clock RNA molecule of all or part of complementation of the messenger RNA(mRNA) of polypeptide of encoding.The overexpression of this antisense rna molecule can cause the expression of natural gene to reduce.Therefore, a plurality of plant that transform with the Antisense Suppression expression cassette are screened to differentiate that those demonstrate the plant that the maximum of expression of polypeptides is round the clock suppressed.
The polynucleotide that are used for Antisense Suppression can be corresponding to all or part of, 5 ' and/or the two complementary sequence all or part of of the encoding sequence of the complementary sequence of 3 ' non-translational region all or part of or the transcript of polypeptide round the clock of encoding and non-translational region of transcript round the clock of the complementary sequence of the sequence of polypeptide round the clock of encoding.In addition, antisense polynucleotides can be complementary with target sequence complete complementary (namely the complementary sequence 100% with target sequence is identical) or part (namely the identity with the complementary sequence of target sequence is lower than 100%).Antisense Suppression also can be used to suppress the expression of the multiple proteins in the same plant.Referring to (for example) U.S. Patent No. 5,942,657.In addition, the part of antisense nucleotide can be used to destroy the expression of target gene.In general, can use at least 50 Nucleotide, 100 Nucleotide, 200 Nucleotide, 300,400,450,500,550 or the sequence of more Nucleotide.The method that suppresses the expression of the native gene in the plant with Antisense Suppression has description: Liu in for example such as Publication about Document and patent, et al., (2002) Plant Physiol.129:1732-1743 (people such as Liu, 2002, " plant physiology ", the 129th volume, 1732-1743 page or leaf), and U.S. Patent No. 5,759,829 and 5,942,657, incorporate by reference each of these reference and patent into this paper.The efficient of Antisense Suppression can by in expression cassette 3 of antisense sequences ' and 5 ' position of polyadenylation signal comprise that the poly-dT district improves.Referring to U.S. Patent Application Publication No.2002/0048814, be incorporated by reference this paper.
Iii. double-stranded RNA disturbs
In some embodiments of the invention, can obtain by double-stranded RNA (dsRNA) interference the round the clock inhibition of expression of polypeptides.Disturb for dsRNA, have adopted RNA molecule (describing for co-suppression as mentioned) and with this have adopted RNA molecule wholly or in part complementary antisense rna molecule at same cells, thereby cause the inhibition of expression of the endogenous messenger RNA(mRNA) of correspondence.
The expression of sense and antisense molecule can be led to will express that box is designed to include simultaneously adopted sequence and antisense sequences is realized.Perhaps, adopted sequence and antisense sequences independent expression cassette can be respectively applied to.Then a plurality of plant that disturb expression cassette to transform with dsRNA are screened to differentiate and demonstrate the plant that the maximum of expression of polypeptides is round the clock suppressed.The method of disturbing to suppress the expression of endogenous plant gene with dsRNA has description: Waterhouse in Publication about Document and patent, et al., (1998) the Proc.Natl.Acad.Sci.USA 95:13959-13964 (people such as Waterhouse, 1998, " institute of NAS periodical ", the 95th volume, the 13959-13964 page or leaf), Liu, et al., (2002) the Plant Physiol.129:1732-1743 (people such as Liu, 2002, " plant physiology ", the 129th volume, the 1732-1743 page or leaf) and WO 99/49029, WO 99/53050, WO 99/61631 and WO00/49035 are incorporated herein by reference each document and patent.
Iv. hairpin RNA disturbs and contains the hairpin RNA interference of intron
In some embodiments of the invention, can disturb by the hairpin RNA (ihpRNA) that hairpin RNA (hpRNA) disturbed or contained intron the inhibition of expression of polypeptides round the clock and obtain.These methods are highly effective suppressing aspect the native gene expression.Referring to Waterhouse and Helliwell, (2003) Nat.Rev.Genet.4:29-38 (Waterhouse and Helliwell,, " genetics is commented on naturally ", the 4th volume, 29-38 page or leaf in 2003) and the reference of wherein quoting.
Disturb for hpRNA, expression cassette is designed to express such RNA molecule, this RNA molecule self hybridization and form the hairpin structure that comprises single-stranded loop district and base pairing stem.Endogenous messenger RNA(mRNA) all or part of that this base pairing stem district comprises the gene that will suppress its expression corresponding to coding has adopted sequence and with this wholly or in part complementary antisense sequences of adopted sequence arranged.Therefore, the base pairing stem district of this molecule has determined the specificity that RNA disturbs usually.HpRNA is very efficient in suppressing the native gene expression, and the RNA that they are induced disturbs and inherited by the plant offspring.Referring to for example Chuang and Meyerowitz, (2000) Proc.Natl.Acad.Sci.USA 97:4985-4990 (Chuang and Meyerowitz,, " institute of NAS periodical ", the 97th volume, 4985-4990 page or leaf in 2000); Stoutjesdijk, et al., (2002) Plant Physiol.129:1723-1731 (people such as Stoutjesdijk, 2002, " plant physiology ", the 129th volume, the 1723-1731 page or leaf) and Waterhouse and Helliwell, (2003) Nat.Rev.Genet.4:29-38 (Waterhouse and Helliwell, 2003, " genetics is commented on naturally ", the 4th volume, 29-38 page or leaf).Disturb to suppress or the method for eliminating genetic expression has description in for example with Publication about Document and patent: Chuang and Meyerowitz with hpRNA, (2000) Proc.Natl.Acad.Sci.USA 97:4985-4990 (Chuang and Meyerowitz, 2000, " institute of NAS periodical ", the 97th volume, the 4985-4990 page or leaf); Stoutjesdijk, et al., (2002) Plant Physiol.129:1723-1731 (people such as Stoutjesdijk,, " plant physiology ", the 129th volume, 1723-1731 page or leaf in 2002); Waterhouse and Helliwell, (2003) Nat.Rev.Genet.4:29-38 (Waterhouse and Helliwell,, " genetics is commented on naturally ", the 4th volume, 29-38 page or leaf in 2003); Pandolfini, et al., the BMC Biotechnology 3:7 (people such as Pandolfini, " BMC biotechnology ", the 3rd volume, the 7th page) and U.S. Patent Application Publication No.2003/0175965, more than each document and patent be incorporated herein by reference.The instantaneous measurement method of the efficient of elimination genetic expression is by Panstruga in the hpRNA construct body, et al., (2003) Mol.Biol.Rep.30:135-140 (people such as Panstruga, 2003, " molecular biology report ", the 30th volume, the 135-140 page or leaf) description (it is incorporated herein by reference).
For ihpRNA, disturbing molecule has the overall structure identical with hpRNA, but this RNA molecule comprises intron in addition, and this intron can be by montage in the cell of expressing this ihpRNA.The use of intron is so that the ring district size in the hairpin RNA molecule minimizes after montage, and this can improve the efficient of interference.Referring to for example Smith, et al., (2000) Nature 407:319-320 (people such as Smith,, " nature ", the 407th volume, 319-320 page or leaf in 2000).In fact, the people such as Smith confirm to use the interference of ihpRNA mediation, and native gene is expressed and is subject to 100% inhibition.Disturb to suppress the method for expression of endogenous plant gene with ihpRNA for example at Smith, et al., (2000) Nature407:319-320 (people such as Smith,, " nature ", the 407th volume, 319-320 page or leaf in 2000); Wesley, et al., (2001) Plant be (people such as Wesley, calendar year 2001, " plant magazine ", the 27th volume, 581-590 page or leaf) J.27:581-590; Wang and Waterhouse, (2001) Curr.Opin.Plant Biol.5:146-150 (Wang and Waterhouse, calendar year 2001, " plant biology is newly seen ", the 5th volume, 146-150 page or leaf); Waterhouse and Helliwell, (2003) Nat.Rev.Genet.4:29-38 (Waterhouse and Helliwell,, " genetics is commented on naturally ", the 4th volume, 29-38 page or leaf in 2003); Helliwell and Waterhouse, (2003) Methods30:289-295 (Helliwell and Waterhouse, 2003, " method ", the 30th volume, the 289-295 page or leaf) describe to some extent and among the U.S. Patent Application Publication No.2003/0180945, more than each document and patent be incorporated herein by reference.
Also can the expression cassette that be used for the hpRNA interference be designed, so that have adopted sequence and antisense sequences not to correspond to endogenous RNA.In this embodiment, this antisense and adopted sequence is arranged at the side of such ring sequence, this ring sequence comprises all or part of nucleotide sequence corresponding to the endogenous messenger RNA(mRNA) of target gene.Thereby, be that the ring district has determined the specificity that RNA disturbs.Referring to for example WO02/00904, incorporate it into this paper by reference.
V. the interference of amplicon mediation
The amplicon expression cassette comprises the sequence of plant-derived virus, and this sequence contains all or part of of target gene but usually do not contain gene whole of this natural viral.This transcription product is present in virus sequence in the transcription product of expression cassette so that can instruct copying of himself.The transcript that is produced by this amplicon can be sense or antisense with respect to target sequence (namely round the clock the messenger RNA(mRNA) of polypeptide).The method that suppresses the expression of endogenous plant gene with amplicon for example has description in Publication about Document and patent: Angell and Baulcombe, (1997) J.16:3675-3684 (Angell and Baulcombe of EMBO, 1997, " European Molecular Bioglogy Organization's proceedings ", the 16th volume, the 3675-3684 page or leaf), Angell and Baulcombe, (1999) Plant is (Angell and Baulcombe, 1999 J.20:357-362, " plant magazine ", the 20th volume, the 357-362 page or leaf) and U.S. Patent No. 6,646,805, more than each document and patent be incorporated herein by reference.
Vi. ribozyme
The polynucleotide of being expressed by expression cassette of the present invention in certain embodiments, are the specific catalytic RNAs of messenger RNA(mRNA) of round the clock polypeptide or have the round the clock specific ribozyme activity of messenger RNA(mRNA) of polypeptide.Thereby these polynucleotide cause the degraded of endogenous messenger RNA(mRNA), thereby cause the round the clock reduction of expression of polypeptides.This method is for example in U.S. Patent No. 4,987, is described in 071, incorporates by reference this patent into this paper.
Vii. siRNA or microRNA
In some embodiments of the invention, the round the clock inhibition of expression of polypeptides can be disturbed through RNA by the gene of expressing coding microRNA (miRNA) and obtain.MiRNA is by about 22 adjusting control agents that ribonucleotide forms, and miRNA is very efficient aspect the expression of inhibition native gene.Referring to for example Javier, et al., (2003) Nature 425:257-263 (people such as Javier,, " nature ", the 425th volume, 257-263 page or leaf in 2003) incorporates the document into this paper by reference.
Disturb for miRNA, expression cassette is designed to express the RNA molecule of miRNAs gene in the imitation.This miRNA genes encoding can form the RNA of hairpin structure, and this hairpin structure contains 22 nucleotide sequences complementary with another native gene (target sequence).Be to suppress Circadian Expression, the sequence of described 22 Nucleotide be selected from transcript sequence round the clock and comprise described round the clock sequence sense orientation 22 Nucleotide and with described 21 Nucleotide that the corresponding antisense sequences of adopted sequence complementation is arranged.The miRNA molecule is very efficient in the expression that suppresses native gene, and the RNA that they are induced disturbs and inherited by the plant offspring.
2. the inhibition based on polypeptide of genetic expression
In one embodiment, the zinc finger protein that described polynucleotide encoding is combined with the gene of polypeptide round the clock of encoding, thus cause the expression of described gene to reduce.In a particular embodiment, this zinc finger protein is bonded to the round the clock control region of gene.In other embodiments, this zinc finger protein is bonded to the messenger RNA(mRNA) of the polypeptide round the clock of encoding and prevents its translation.Selection by the method in the site of zinc finger protein target for example in U.S. Patent No. 6,453,242 are described, the method of utilizing zinc finger protein to suppress the genetic expression in the plant for example is described in U.S. Patent Application Publication No.2003/0037355, and each incorporates this paper by reference into these patents.
3. the inhibition based on polypeptide of protein active
In some embodiments of the invention, the antibody that described polynucleotide encoding is such, at least a round the clock polypeptide of this antibodies also reduces this round the clock activity of polypeptide.In another embodiment, the combination of antibody causes the round the clock turnover of mixture of the antibody that undertaken by the cell Quality Control Mechanism to increase.Antibody in vegetable cell expression and by antibody expression and be bonded to protein in the vegetable cell to come the Inhibitory molecules approach be known in the art.Referring to for example Conrad and Sonnewald, (2003) Nature Biotech.21:35-36 (Conrad and Sonnewald,, " Nature Biotechnol ", the 21st volume, 35-36 page or leaf in 2003), it is incorporated herein by reference.
4. gene disruption
In some embodiments of the invention, by the destruction round the clock gene of polypeptide of encoding, reduce or eliminate the round the clock activity of polypeptide.Can destroy the round the clock gene of polypeptide of encoding by any method known in the art.For example, in one embodiment, destroy described gene by transposon tagging.In another embodiment, by utilizing random mutagenesis or directed mutagenesis to come the plant that plant carries out mutagenic treatment and selects to have the cell quantity regulatory factor activity of reduction is destroyed described gene.
I. transposon tagging
In one embodiment of the invention, use transposon tagging to reduce or eliminate the activity of one or more round the clock polypeptide.Transposon tagging is included in and inserts transposon in the endogenous round the clock gene to reduce or to eliminate the expression of described round the clock polypeptide." round the clock gene " gene according to round the clock polypeptide of the present invention that means to encode.
In this embodiment, reduce or eliminate one or more round the clock expression of polypeptide by in the control region of the gene of polypeptide round the clock of encoding or coding region, inserting transposon.Be in the exon, intron, 5 of gene round the clock ' or 3 ' non-translated sequence, promotor or any other regulating and controlling sequence in transposon can be used for reducing or eliminating expression and/or the activity of coded round the clock polypeptide.
The method of carrying out transposon tagging for the specific gene to plant is known in the art.Referring to for example Maes, et al., (1999) Trends Plant Sci.4:90-96 (people such as Maes,, " plant science trend ", the 4th volume, 90-96 page or leaf in 1999); Dharmapuri and Sonti, (1999) FEMS Microbiol.Lett.179:53-59 (Dharmapuri and Sonti,, " federation of European Microbiological Societies's microbiology wall bulletin ", the 179th volume, 53-59 page or leaf in 1999); Meissner, et al., (2000) Plant be (people such as Meissner,, " plant magazine ", the 22nd volume, 265-274 page or leaf in 2000) J.22:265-274; Phogat, et al., (2000) J.Biosci.25:57-63 (people such as Phogat,, " bio-science magazine ", the 25th volume, 57-63 page or leaf in 2000); Walbot, (2000) Curr.Opin.Plant Biol.2:103-107 (Walbot,, " plant biology is newly seen ", the 2nd volume, 103-107 page or leaf in 2000); Gai, et al., (2000) Nucleic Acids Res.28:94-96 (people such as Gai,, " nucleic acids research ", the 28th volume, 94-96 page or leaf in 2000); Fitzmaurice, et al., (1999) Genetics 153:1919-1928 (people such as Fitzmaurice,, " genetics ", the 153rd volume, 1919-1928 page or leaf in 1999)).In addition, at Bensen, et al., (1995) Plant Cell 7:75-84 (people such as Bensen, nineteen ninety-five, " vegetable cell ", the 7th volume, 75-84 page or leaf); Mena, et al., (1996) the Science 274:1537-1540 (people such as Mena, 1996, " science ", the 274th volume, the 1537-1540 page or leaf) and U.S. Patent No. 5, the TUSC method of selecting Mu to insert in the gene of selecting has been described in 962,764, more than each document and patent incorporate by reference this paper into.
Ii. the mutant plant of activity decreased
For reducing or the other method of eliminating the expression of the native gene in the plant also be known in the art, and can be applied to similarly the present invention.These methods comprise other forms of mutagenesis, ethyl methane sulfonate mutagenesis, deletion mutagenesis and the fast neutron deletion mutagenesis of inducing for example, and the fast neutron deletion mutagenesis is used for differentiating the plant that native gene has wherein lacked in reverse genetics mode (using PCR).Example such as these methods of need sees also Ohshima, et al., (1998) Virology 243:472-481 (people such as Ohshima,, " virusology ", the 243rd volume, 472-481 page or leaf in 1998); Okubara, et al., (1994) Genetics 137:867-874 (people such as Okubara, 1994, " genetics ", the 137th volume, the 867-874 page or leaf) and Quesada, et al., (2000) Genetics 154:421-436 (people such as Quesada, 2000, " genetics ", the 154th volume, 421-436 page or leaf), each document is incorporated this paper by reference into more than.In addition, a kind of quick and automatable method TILLING (Targeting Induced Local Lesions In Genomes (directional induction genome local sudden change) of the sudden change for the screening chemical induction) also be applicable to the present invention, the method is utilized sex change HPLC or to the selectivity endonuclease digestion of selected PCR product.Referring to McCallum, et al., (2000) Nat.Biotechnol.18:455-457 (people such as McCallum,, " Nature Biotechnol ", the 18th volume, 455-457 page or leaf in 2000), it incorporates this paper by reference into.
It is known in the art affecting genetic expression or disturbing the sudden change of the function of coded protein.Insertion mutation in the gene extron causes null mutant usually.The sudden change of conserved residues is being effective especially aspect the cell quantity regulatory factor activity of the coded protein of inhibition.Plant round the clock polypeptide be suitable for that to eliminate cell quantity regulatory factor activity be that the conserved residues that target is carried out mutagenesis has obtained describing.Can separate this mutant according to the program of knowing, and can by genetic cross to difference the round the clock sudden change in the locus carry out stacking.Referring to for example Gruis, et al., (2002) Plant Cell 14:2863-2882 (people such as Gruis,, " vegetable cell ", the 14th volume, 2863-2882 page or leaf in 2002).
In another embodiment of the present invention, dominant mutant is because the restructuring of gene inversion and duplicate loci can be used for causing the RNA silence.Referring to for example Kusaba, et al., (2003) Plant Cell15:1455-1467 (people such as Kusaba,, " vegetable cell ", the 15th volume, 1455-1467 page or leaf in 2003).
The present invention contain other for reducing or eliminate one or more round the clock methods of the activity of polypeptide.Be used for to change or the example of the additive method of the genome nucleotide sequence of mutant plant is known in the art, include but not limited to use RNA:DNA carrier, RNA:DNA mutational vector, RNA:DNA repair vector, mix double chain oligonucleotide, from complementary RNA: the few nuclear of DNA oligonucleotide and recombined engineering base (recombinogenic oligonucleobase).This carrier and using method are known in the art.Referring to for example U.S. Patent No. 5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972 and 5,871,984, more than each patent incorporate by reference this paper into.In addition referring to WO 98/49350, WO 99/07865, WO 99/25821 and Beetham, et al., (1999) the Proc.Natl.Acad.Sci.USA 96:8774-8778 (people such as Beetham, 1999, " institute of NAS periodical ", the 96th volume, 8774-8778 page or leaf), each document and patent are incorporated this paper by reference into more than.
Iii. it is active to regulate Growth of Cells and/or allelotaxis
In specific method, by level or active level and/or the activity that increases tissue development in the plant that increases the round the clock polypeptide in the plant.The level and/or the active method that increase the round the clock polypeptide in the plant are discussed at this paper elsewhere.In brief, this method comprises provides round the clock polypeptide of the present invention to plant, thereby increases this round the clock level and/or activity of polypeptide.In other embodiments, can be by the round the clock round the clock nucleotide sequence of polypeptide of encoding be provided like this: in plant, introduce the polynucleotide that comprise round the clock nucleotide sequence of the present invention, express this round the clock sequence, increase this round the clock activity of polypeptide, and therefore increase the histiocytic number in plant or the plant part.In other embodiments, the round the clock constructs in the introduced plant is by in the stable genome that is incorporated into plant.
In additive method, by level and/or active cell number and the biomass that increases plant tissue that increases the round the clock polypeptide in the plant.This method has been carried out open in detail at this paper elsewhere.In such method, nucleotide sequence is introduced in the plant round the clock, thereby the expression of described round the clock nucleotide sequence can reduce the active of polypeptide round the clock and increase plant-growth and/or allelotaxis in plant or the plant part.In other embodiments, the round the clock constructs in the introduced plant is by in the stable genome that is incorporated into plant.
As discussed above, the technician will appreciate that, suitable promotor can be used for the plant-growth of regulating plant and/or the levels/activity of allelotaxis's polynucleotide and polypeptide.The exemplary promotor of this embodiment is disclosed at this paper elsewhere.
Therefore, the present invention also provides when have the plant-growth of change and/or allelotaxis's plant relatively the time with the plant-growth of control plant tissue and/or allelotaxis.In one embodiment, plant of the present invention has the levels/activity of the round the clock polypeptide of the present invention of increase, thereby has plant-growth and/or the allelotaxis of increase in plant tissue.In other embodiments, plant of the present invention has the level of the round the clock polypeptide of the present invention that reduces or eliminate, thereby has plant-growth and/or the allelotaxis of reduction in plant tissue.In other embodiments, this kind of plant in its genome stable integration comprise the nucleic acid molecule of round the clock nucleotide sequence of the present invention, this sequence effectively is connected to the promotor that can drive the expression in this vegetable cell.
Iv. regulate root development
The method that is used for the root development of regulating plant is provided.So-called " adjusting root development " means any change of plant development of root when comparing with control plant.This change of root development includes but not limited to: the degree that nascent Rate of root growth, root fresh weight, lateral root and adventive root form, vascular system, meristematic tissue are grown or the radial dilatation degree.
The method that is used for the root development of regulating plant is provided.Described method comprises regulates this round the clock level and/or the activity of polypeptide in plant.In one approach, round the clock polypeptide of the present invention is offered plant.In another approach, by round the clock nucleotide sequence is provided like this: will comprise in the polynucleotide introduced plant of round the clock nucleotide sequence of the present invention, express this round the clock sequence, thus the change root development.In other other method, the round the clock constructs in the introduced plant is by in the stable genome that is incorporated into plant.
In additive method, regulate root development by changing round the clock level or the activity of polypeptide in plant.The increase of activity can cause at least a or multiple following change to root development round the clock when with the control plant comparison, includes but not limited to: the radial dilatation that larger root meristematic tissue, root growth increase, strengthen, the vascular system of enhancing, the root branch of increase, more adventive root and/or the heavy increase of bright root.
" root growth " used herein contains in monocotyledons and the dicotyledons different piece that consists of root system aspect all of the growth of the different steps of root system development.Should be appreciated that root growth strengthens to be strengthened by one or more the growth in its each several part (comprising primary root, lateral root, adventive root etc.) causes.
The method of measuring this growth change in the root system is known in the art.Referring to for example U.S. Patent Application Publication No.2003/0074698 and Werner, et al., (2001) the PNAS 18:10487-10492 (people such as Werner, calendar year 2001, " institute of NAS periodical ", the 18th volume, the 10487-10492 page or leaf), incorporate by reference these two pieces of documents into this paper.
As discussed above, the technician will be familiar with the suitable promotor for the root development of regulating plant.The exemplary promotor that is used for this embodiment comprises constitutive promoter and the preferred promotor of root.The preferred promotor of exemplary root is open in this paper other places.
The weight that activity by increasing polypeptide round the clock and/or level stimulate root growth and increase root also is applied aspect the lodging resistance of plant improving.Term " lodging resistance " or " lodging resistance " refer to that plant makes the ability that himself is fixed to soil.For having the plant of erectting or partly erectting habit, this term also refer under unfavorable (environment) condition, to be kept upright ability of position.This proterties relates to size, the degree of depth and the form of root system.In addition, level and/or active root growth and the increase root weight of stimulating of polypeptide also are applied in the external proliferation that promotes explant by increasing round the clock.
In addition, since the round the clock active level that increases and/or the active higher root biomass that causes output is had direct effect and the generation by the compound of the cell cultures deposits yields of root cells or transgenosis root cells or described transgenosis root cells is had indirectly impact.An example of the concern compound that produces in root culturd is Shikonin (shikonin), and its output can advantageously strengthen by described method.
The plant that has modulated root development when therefore, the present invention also provides and compared with the root development of control plant.In certain embodiments, plant of the present invention has a levels/activity of the round the clock polypeptide of the present invention that has improved and has root growth and/or the root biomass that has strengthened.In other embodiments, this kind of plant in its genome stable integration comprise the nucleic acid molecule of round the clock nucleotide sequence of the present invention, this sequence effectively is connected to the promotor that can drive the expression in this vegetable cell.
V. regulate seedling and leaf development
Also provide and be used for the seedling of regulating plant and the method for leaf development.So-called " regulating seedling and/or leaf development " it means any change of the growth of plantling and/or leaf.This change in seedling and/or the leaf development includes but not limited in seedling branch growing tissue's development, in the change aspect number of sheets order, leaf size, leaf and stem vascular system, panel length and the leaf aging." leaf development " used herein and " seedling growth " is encompassed in and consists of respectively leaf system in monocotyledons and the dicotyledons and unify aspect all of the growth of different piece in these phylogenetic different stepss of seedling system.The method of measuring this growth change in seedling and the leaf system system is known in the art.Referring to for example Werner, et al., (2001) the PNAS 98:10487-10492 (people such as Werner, calendar year 2001, " institute of NAS periodical ", the 98th volume, the 10487-10492 page or leaf) and U.S. Patent Application Publication No.2003/0074698, each incorporates this paper by reference into these two pieces of documents.
The method of seedling and/or leaf development comprises activity and/or the level of regulating round the clock polypeptide of the present invention in the regulating plant.In one embodiment, provide round the clock sequence of the present invention.In other embodiments, can provide as getting off this round the clock nucleotide sequence: will comprise in the polynucleotide introduced plant of round the clock nucleotide sequence of the present invention, express this round the clock sequence, thus change seedling and/or leaf development.In other embodiments, the round the clock constructs in the introduced plant is by in the stable genome that is incorporated into plant.
In specific embodiment, regulate seedling or leaf development by the level and/or the activity that reduce the round the clock polypeptide in the plant.When with control plant relatively the time, round the clock active reduction can cause the change of at least a or multiple following seedling and/or leaf development, includes but not limited to: number of sheets order reduces, leaf surface reduces, the dimension pipe reduces, internode is short and grow short and small and leaf is aging delays.
As discussed above, the technician will recognize that the suitable promotor for seedling and the leaf development of regulating plant.The exemplary promotor that is used for this embodiment comprises constitutive promoter, the preferred promotor of seedling, the preferred promotor of seedling branch growing tissue and the preferred promotor of leaf.Exemplary promotor is open in this paper other places.
Reducing round the clock activity in the plant and/or level can cause internode short and grow short and small.Thereby method of the present invention can have application aspect the generation plant of short stem.In addition, as discussed above, the two the growth of the round the clock active adjustable root section of adjusting in the plant and seedling.Thereby the present invention also provides the method that is used for changing root/seedling ratio.Can further regulate seedling or leaf development by the level and/or the activity that reduce the round the clock polypeptide in the plant.
The plant that has modulated seedling and/or leaf development when therefore, the present invention also provides and compared with control plant.In certain embodiments, plant of the present invention has the levels/activity of the round the clock polypeptide of the present invention that has improved, and has changed the growth of seedling and/or leaf.This change includes but not limited to compare with control plant, and number of sheets order increases, leaf surface increases, vasculature increases, internode is longer and the plant plant height increases and leaf is aging changes.In other embodiments, plant of the present invention has the levels/activity of the round the clock polypeptide of the present invention that has reduced.
Vi regulates germinal tissue and grows
Provide and be used for regulating the method that germinal tissue is grown.In one embodiment, provide the method that is used for the flower development of regulating plant.So-called " adjusting flower development " mean with wherein round the clock the activity of polypeptide or level also not modulated control plant compare any change of the structure of the germinal tissue of plant." adjusting flower development " also comprise with wherein round the clock the not modulated control plant of the activity of polypeptide or level compare any change in the moment of plant reproductive tissue development (being flower development delay or in advance constantly).The following variation of macroscopic view change can be included in environment-stress the time: the size of reproductive organ, shape, number or position, in the development time cycle that these structures form, perhaps keep or develop ability through the process of blooming.Microcosmic changes the change of type or the shape that can comprise the cell that consists of reproductive organ.
The method of the flower development in the regulating plant comprises the round the clock activity in the regulating plant.In a method, provide round the clock sequence of the present invention.Can be by round the clock nucleotide sequence be provided like this: will comprise in the polynucleotide introduced plant of round the clock nucleotide sequence of the present invention, express this round the clock sequence, thus the growth of change flower.In other embodiments, the round the clock constructs in the introduced plant is by in the stable genome that is incorporated into plant.
In concrete method, regulate flower development by the level or the activity that reduce the round the clock polypeptide in the plant.When comparing with control plant, round the clock active reduction can cause at least a or multiple following flower development to change, and includes but not limited to: the delay of blooming, flower reduced number, part male sterile and the minimizing of setting seeds.Induced flowering postpones or suppresses to bloom to can be used for strengthening the output of the fodder crop such as clover.The method that is used for this growth change of measurement flower development is known in the art.Referring to for example Mouradov, et al., (2002) The Plant Cell S111-S130 (people such as Mouradov,, " vegetable cell ", S111-S130 page or leaf in 2002) incorporates the document into this paper by reference.
As discussed above, the technician will be familiar with the suitable promotor for the flower development of regulating plant.The exemplary promotor that is used for this embodiment comprises constitutive promoter, inducible promoter, the preferred promotor of seedling, the preferred promotor of inflorescence.
In additive method, regulate flower development by the level and/or the activity that increase round the clock sequence of the present invention.Thereby this method can comprise round the clock nucleotide sequence introducing plant is increased the round the clock activity of polypeptide.In additive method, the round the clock constructs in the introduced plant is by in the stable genome that is incorporated into plant.The expression that increases round the clock sequence of the present invention can be regulated the flower development during coercing.This method is described in this paper other places.Therefore, the present invention also provides with the flower development of control plant and has compared the plant with modulated flower development.Composition comprises the levels/activity of the round the clock polypeptide of the present invention with increase and has the plant of the flower development of change.Composition also comprises the plant of the levels/activity of the round the clock polypeptide of the present invention with increase, and wherein said plant keeps when coercing or continues the process of blooming.
The method that increases seed size and/or weight with round the clock sequence of the present invention also is provided.The method comprises and improves for example activity of the round the clock sequence in the seed of plant or plant part.The increase of seed sizes and/or weight comprises that seed sizes or weight increase and/or size or the weight of one or more kinds of subdivisions (comprising for example embryo, endosperm, kind skin, aleuron or cotyledon) increase.
As discussed above, the technician will recognize the suitable promotor for increasing seed sizes and/or seed weight.The exemplary promotor of this embodiment comprises the preferred promotor of promotor, embryo and the preferred promotor of endosperm of constitutive promoter, inducible promoter, select seeds.
Seed sizes in the reduction plant and/or the method for seed weight comprise the round the clock activity that reduces in this plant.In one embodiment, can provide as getting off this round the clock nucleotide sequence: will comprise in the polynucleotide introduced plant of round the clock nucleotide sequence of the present invention, and express this round the clock sequence, thereby reduce seed weight and/or size.In other embodiments, the round the clock constructs in the introduced plant is by in the stable genome that is incorporated into plant.
Also recognize, increase seed sizes and/or weight and can also be attended by the increase of the seedling speed of growth or the increase of early stage vigor.As used herein, term " early stage vigor " refer to plant in early days between the growth period ability of Fast Growth and relate to sprouting after successful planting, have well-developed root system and well-developed photosynthesis organ.In addition, when when comparing, the increase of seed sizes and/or weight also can cause the increase of plant biomass.
Therefore, the present invention also provides and had the seed weight of increase and/or the plant of seed sizes when with the control plant comparison.In other embodiments, vigor with increase and the plant of plant biomass are also provided.In certain embodiments, plant of the present invention has a levels/activity of the round the clock polypeptide of the present invention that has improved and has seed weight and/or the seed sizes that has increased.In other embodiments, this kind of plant in its genome stable integration comprise the nucleic acid molecule of round the clock nucleotide sequence of the present invention, this sequence effectively is connected to the promotor that can drive the expression in this vegetable cell.
Vii. the using method of promotor polynucleotide round the clock
When with DNA construct assembling so that when promoter sequence effectively is connected to the nucleotide sequence that comprises the polynucleotide of paying close attention to round the clock, the polynucleotide that comprise disclosed round the clock promotor among the present invention with and variant and fragment can be used for the genetic manipulation of any host cell (preferred plant cell).Like this, round the clock promotor polynucleotide of the present invention are provided for expressing in the host cell of paying close attention in expression cassette together with the polynucleotide sequence of paying close attention to.As described in " example " of the present invention part, round the clock promoter sequence of the present invention is expressed in Various Tissues, so this promoter sequence can be used for regulating and control time and/or the space expression of the polynucleotide of paying close attention to.
Synthetic hybrid promoter district is known in the art.This district inclusion effectively is connected to the upstream promoter element of polynucleotide of the promoter element of another polynucleotide.In one embodiment of the invention, express by synthetic hybrid promoter control heterologous sequence, this synthetic hybrid promoter comprises round the clock promoter sequence of the present invention or its variant or the fragment that effectively is connected to from the upstream promoter element of allogeneic promoter.It is identified and can be used for producing synthetic promoter to relate to the upstream promoter element of plant defense system.Referring to for example Rushton, et al., (1998) Curr.Opin.Plant Biol.1:311-315 (people such as Rushton,, " plant biology is newly seen ", the 1st volume, 311-315 page or leaf in 1998).Perhaps, synthetic round the clock promoter sequence can comprise the round the clock repetition of the interior upstream promoter element that exists of promoter sequence.
Have realized that the round the clock encoding sequence that promoter sequence of the present invention can be natural with it uses.The DNA construct that comprises the round the clock promotor that effectively is connected with its natural round the clock gene can be used for transforming any plant of paying close attention to and causes that required phenotype changes, and for example regulates cell number, regulates any other phenotype that root, seedling, leaf, flower and Embryo development, stress tolerance and this paper elsewhere are described.
Promotor nucleotide sequence disclosed herein and method can be used for regulating and control the expression of any heterologous nucleotide sequence in host plant in order to change plant phenotype.There is multiple phenotype to change and merits attention, comprise that the lipid acid in the modified plant forms, change the aminoacids content of plant, pathogenic agent defense mechanism of change plant etc.These results can realize by expressing heterologous product in plant or the expression that increases endogenous product.Perhaps, these results can realize by the expression that reduces one or more endogenous products (particularly enzyme or cofactor) in plant.These changes cause the phenotype of conversion of plant to change.
The gene of paying close attention to has reflected the participant's of crop exploitation commercial market and interests.The crop of paying close attention to and market changing, and along with developing country has opened world market, also new crop and technology will appear.In addition, along with our increase to agronomy character and characteristic such as output and heterotic understanding, will respective change to the selection of the gene that be used for to transform.The general categories of the gene of paying close attention to comprises those genes (referring to such as zinc), those genes (such as kinases) that relate to communication that for example relate to information and relates to special those genes (such as heat shock protein(HSP)).Genetically modified more specifically classification for example comprises that coding is to the gene of the important proterties of agronomy, insect-resistant, disease resistance, Herbicid resistant, sterility, grain feature and commerical prod.Generally speaking, the gene of paying close attention to comprise relate to grease, starch, carbohydrate or nutrient metabolism those and affect those of seed size, sucrose carrying capacity etc.
In certain embodiments, nucleotide sequence of the present invention and other polynucleotide sequence associatings (" stacking ") of paying close attention to can be used, in order to produce the plant with desired phenotype.The combination that generates can comprise any one or many persons' a plurality of copies in the polynucleotide paid close attention to.Polynucleotide of the present invention can have with the combination stacked of any gene or gene the plant of multiple required proterties combination with generation, described proterties include but not limited to the required proterties of animal-feed for example high oil base because (for example U.S. Patent No. 6,232,529); The amino acid of balance (hordothionins (U.S. Patent No. 5,990,389 for example; 5,885,801; 5,885,802 and 5,703,409); Barley high-lysine (Williamson, et al., (1987) Eur.J.Biochem.165:99-106 (people such as Williamson, 1987, " european journal of biological chemistry ", the 165th volume, 99-106 page or leaf) and WO 98/20122) and homomethionine protein (Pedersen, et al., (1986) J.Biol.Chem.261:6279 (people such as Pedersen,, " journal of biological chemistry " in 1986, the 261st volume, the 6279th page); Kirihara, et al., (1988) Gene 71:359 (people such as Kirihara, 1988, " gene ", the 71st volume, the 359th page) and Musumura, et al., (1989) Plant Mol.Biol.12:123 (people such as Musumura,, " molecular biology of plants " in 1989, the 12nd volume, the 123rd page)); The digestibility that has improved (modified storage protein (the U.S. Patent application No.10/053 that submits to calendar year 2001 November 7 for example, 410) and Trx (the U.S. Patent application No.10/005 that submits to December 3 calendar year 2001,429)), incorporate by reference above-mentioned disclosure into this paper.Polynucleotide of the present invention also can be stacking with proterties pest-resistant, disease-resistant or that antiweed is required (bacillus thuringiensis toxic protein (U.S. Patent No. 5,366,892 for example; 5,747,450; 5,737,514; 5723,756; 5,593,881; Geiser, et al., (1986) Gene 48:109 (people such as Geiser,, " gene ", the 48th volume, the 109th page in 1986)); Lectin (Van Damme, et al., (1994) Plant Mol.Biol.24:825 (people such as Van Damme,, " molecular biology of plants ", the 24th volume, the 825th page in 1994)); Fumonisin detoxification genes (U.S. Patent No. 5,792,931); Nontoxic gene and disease-resistant gene (Jones, et al., (1994) Science 266:789 (people such as Jones,, " science ", the 266th volume, the 789th page in 1994); Martin, et al., (1993) Science 262:1432 (people such as Martin,, " science ", the 262nd volume, the 1432nd page in 1993); Mindrinos, et al., (1994) Cell 78:1089 (people such as Mindrinos,, " cell ", the 78th volume, the 1089th page in 1994)); Acetolactate synthestase (ALS) mutant that causes Herbicid resistant, for example S4 and/or Hra sudden change; Glutamine synthetase inhibitor is glufosinates or basta (for example bar gene) and glyphosate resistance (EPSPS gene) for example) and the required proterties of processing or treating product high oil (for example U.S. Patent No. 6,232,529) for example; Modified oil (fatty acid desaturase gene (U.S. Patent No. 5,952,544 for example; WO 94/11516)); (for example U.S. Patent No. 5 for treated starch (for example ADPG pyrophosphorylase (AGPase), amylosynthease (SS), Q-enzyme (SBE) and starch debranching enzyme (SDBE)) and polymkeric substance or biological plastics, 602,321; β-ketothiolase, poly hydroxybutyric acid synthetic enzyme and Acetoacetyl-CoA reductase (Schubert, et al., (1988) J.Bacteriol.170:5837-5847 (people such as Schubert, 1988, " bacteriology magazine ", the 170th volume, 5837-5847 page or leaf)) be conducive to the expression of polyhydroxyalkanoatefrom (PHA)), above-mentioned disclosure is incorporated this paper by reference into.Can also and affect for example male sterile (for example referring to U.S. Patent No. 5 with polynucleotide of the present invention, 583,210), the economical character of straw stiffness, flowering time and so on or for example cell cycle regulating or gene target (for example WO 99/61619; WO 00/17364; WO 99/25821) and so on the polynucleotide combination of transformation technology proterties, above disclosure is integrated with this paper by reference.
In one embodiment, the sequence of paying close attention to can be improved plant-growth and/or crop yield.For example, the sequence of paying close attention to comprises important gene on the agronomy that can cause Primary Root System or the improvement of lateral root system.This genoid includes but not limited to nutritive substance/water translocator and growth inducing.The example of this genoid includes but not limited to corn plasma membrane H +ATP enzyme (MHA2) (Frias, et al., (1996) Plant Cell 8:1533-44 (people such as Frias,, " vegetable cell ", the 8th volume, 1533-1544 page or leaf in 1996)); AKT1, the i.e. component of potassium picked-up tissue in the Arabidopis thaliana (Spalding, et al., (1999) J Gen Physiol113:909-18 (people such as Spalding,, " general physiology magazine ", the 113rd volume, 909-918 page or leaf in 1999)); The RML gene, it is activating cells mitotic cycle (Cheng, et al., (1995) Plant Physiol 108:881 (people such as Cheng, nineteen ninety-five, " plant physiology ", the 108th volume, the 881st page)) in root-tip cells; Maize glutamine synthetase gene (Sukanya, et al., (1994) Plant Mol Biol 26:1935-46 (people such as Sukanya, 1994, " molecular biology of plants ", the 26th volume, 1935-1946 page or leaf)) and oxyphorase (Duff, et al., (1997) J.Biol.Chem27:16749-16752 (people such as Duff,, " journal of biological chemistry " in 1997, the 27th volume, the 16749-16752 page or leaf); Arredondo-Peter, et al., (1997) Plant Physiol.115:1259-1266 (people such as Arredondo-Peter,, " plant physiology ", the 115th volume, 1259-1266 page or leaf in 1997); Arredondo-Peter, et al., the reference that (1997) Plant Physiol 114:493-500 (people such as Arredondo-Peter,, " plant physiology ", the 114th volume, 493-500 page or leaf in 1997) and this paper quote).The sequence of paying close attention to also can be used for expressing the gene antisense nucleotide sequence of negative impact root development.
In addition, except utilizing traditional breeding method, but important proterties on the agronomy hereditary change such as grease, starch and the protein content also.Modification comprises the content, the level that increases Methionin or sulphur that increase oleic acid, saturated or unsaturated oil, indispensable amino acid and Modified Starch is provided.U.S. Patent No. 5,703, it is protein modified to have described Hordothionin in 049,5,885,801,5,885,802 and 5,990,389, incorporates by reference these documents into this paper.Another example is U.S. Patent No. 5,850, rich Methionin and/or rich sulphur Seed Storage Protein by soybean 2S albumin coding described in 016, and Williamson, et al., (1987) Eur.J.Biochem.165:99-106 (people such as Williamson, 1987, " european journal of biological chemistry ", the 165th volume, the 99-106 page or leaf) the described chymotrypsin inhibitor that is derived from barley, above disclosure is incorporated this paper by reference into.
Can increase the level of preliminary election amino acid in coded polypeptide by the derivative that site-directed mutagenesis produces encoding sequence.For example, the gene (BHL) of coding barley high-lysine polypeptide comes from the barley chymotrypsin inhibitor, be illustrated in the U.S. Patent application No.08/740 that submitted on November 1st, 1996,682 and WO 98/20133, incorporate by reference the disclosure of these two pieces of documents into this paper.Other protein comprise that the plant protein that is rich in methionine(Met) is for example from sunflower seed (Lilley, et al., (1989) Proceedings of the World Congress on Vegetable Protein Utilization in Human Foods and Animal Feedstuffs, ed.Applewhite (American Oil Chemists Society, Champaign, Illinois), pp.497-502 (the people such as Lilley, 1989, " about the world convention collection of thesis of vegetable-protein utilization in human foods and the animal-feed ", Applewhite edits (association of U.S. oil chemistry man, Illinois champagne), the 497-502 page or leaf), it is incorporated herein by reference); Corn (Pedersen, et al., (1986) J.Biol.Chem.261:6279 (people such as Pedersen,, " journal of biological chemistry ", the 261st volume, the 6279th page in 1986); Kirihara, et al., (1988) Gene 71:359 (people such as Kirihara, 1988, " gene ", the 71st volume, the 359th page), these two pieces of documents all are incorporated herein by reference) and paddy rice (Musumura, et al., (1989) the Plant Mol.Biol.12:123 (people such as Musumura, 1989, " molecular biology of plants ", the 12nd volume, the 123rd page), it is incorporated herein by reference).Important genes encoding latex, Floury 2, somatomedin, the storage of seeds factor and transcription factor on other agronomy.
Insect-resistant gene codified is to the resistance of the insect that has a strong impact on output, described insect such as rootworm, cutworm, European corn borer etc.This class is drawn together for example bacillus thuringiensis toxic protein plasmagene (U.S. Patent No. 5,366,892; 5,747,450; 5,736,514; 5,723,756; 5,593,881 and Geiser, et al., (1986) Gene 48:109 (people such as Geiser,, " gene ", the 48th volume, the 109th page in 1986)) etc.
The gene of the disease-resistant proterties of encoding comprises detoxification genes, for example resists the gene (U.S. Patent No. 5,792,931) of fumonisin; Nontoxic (avr) and disease resistance (R) gene (Jones, et al., (1994) Science 266:789 (people such as Jones,, " science ", the 266th volume, the 789th page in 1994); Martin, et al., (1993) Science 262:1432 (people such as Martin, 1993, " science ", the 262nd volume, the 1432nd page) and Mindrinos, et al., (1994) Cell 78:1089 (people such as Mindrinos,, " cell " in 1994, the 78th volume, the 1089th page)) etc.
The Herbicid resistant proterties can comprise that coding (for example contains the sudden change that causes this resistance for the gene of the resistance that plays the weedicide (particularly sulfonylurea herbicide) that suppresses acetolactate synthase (ALS) effect, acetolactate synthase (ALS) gene of S4 and/or Hra sudden change particularly), coding is for the gene (for example bar gene) of the resistance that plays the weedicide that suppresses the glutamine synthase effect such as glufosinates or basta, or other these genoids known in the art.The bar genes encoding is for the resistance of weedicide basta, and the nptII genes encoding is for the resistance of microbiotic kantlex and Geneticin, and als gene sudden change coding is for the resistance of chlorsulfuron.
Also codified is in expression cassette for sterile gene, and castrating for physics provides alternative means.The example of the gene that can this class mode uses comprises the preferred gene of male tissue and gene such as QM with male sterile phenotype, and it is in U.S. Patent No. 5,583, is described in 210.Other genes comprise that kinases and coding grow those of poisonous compound to male or female gamete body.
The quality of grain is reflected in such as following proterties: the quality of the content of saturated and unsaturated oil and type, indispensable amino acid and quantity, cellulosic content.In corn, the hordothionin albumen of modification is in U.S. Patent No. 5,703, is described in 049,5,885,801,5,885,802 and 5,990,389.
Also can be in the commercial proterties of gene coding, described gene can increase the starch that for example is used for alcohol production, or protein expression is provided.The important commercial use of another of conversion of plant is to produce polymkeric substance and biological plastics, as in U.S. Patent No. 5,602, describes in 321.Such as beta-keto thiolase, PHB enzyme (polyhydroxybutyrate synthase) and Acetoacetyl-CoA reductase (referring to Schubert, et al., (1988) J.Bacteriol.170:5837-5847 (people such as Schubert, 1988, " bacteriology magazine ", the 170th volume, 5837-5847 page or leaf)) be conducive to the expression of polyhydroxyalkanoatefrom (PHA).
The external source product comprises plant enzyme and product and from those of other sources that comprise prokaryotic organism and other eukaryotes.This class product comprises enzyme, cofactor, hormone etc.Can increase protein, the amino acids distribution that particularly has an improvement is with the level of the modifying protein that improves Plant Nutritional Value.This can realize by this proteinoid that expression has an aminoacids content of raising.
Viii. the evaluation of extra cis-acting elements
Can use the extra cis-acting elements of multiple standards technical evaluation round the clock promotor disclosed herein, described standard technique comprises for example nucleotide deletion analysis, namely deletes one or more Nucleotide and analyzes regulation activity from 5 of promotor ' end or inside; Carry out the dna binding protein dna analysis with DNA enzyme I footprinting; Interference methylates; Electrophoretic mobility shift assay; Carry out vivo gene group footprint analysis and other routine analyses with the PCR that connects mediation, perhaps by carrying out the dna sequence dna similarity analysis with conventional dna sequence dna comparative approach and other known cis element motifs, and by statistical method for example hidden Markov model (HMM) identify.Can further analyze cis element with the mutation analysis of one or more Nucleotide or by other ordinary methods.
Ix. chimeric promoters
The chimeric promoters that makes up one or more cis elements is known (referring to Venter, et al., (2008), Trends in Plant Science, 12 (3): 118-124 (people such as Venter, 2008, " plant science trend ", the 12nd volume, the 3rd phase, 118-124 page or leaf)).Comprise from the chimeric promoters of the cis element of promotor disclosed herein and flanking sequence thereof and can be other promotors, for example tissue-specific promotor by engineered.Second promoter fragment that for example, can be fused to by comprising first promoter fragment from incitant (round the clock) the page element of a promotor incitant (tissue specificity) cis element that comprises from another promotor produces chimeric promoters; The chimeric promoters of gained can increase the genetic expression of the transcribed polynucleotide molecule of connection round the clock with the mode of being connected.Controlling element disclosed herein is used for engineered chimeric promoters, for example by this element being placed the upstream of minimal promoter.
Can understand better the present invention with reference to following limiting examples.It will be understood to those of skill in the art that and to implement other embodiment of the present invention in the situation of spirit and scope of invention of claims protections not breaking away from disclosed herein and be subjected to.
Example
Diel rhythm research in example 1. corns
Milpa (B73 genotype) is planted under the field condition and in the V14-15 stage of reproduction samples.According to the record (materials and methods) of U.S. Navy Observatory, the illumination condition during sampling is about 14.75 hours daylight.Since the 1st day sunrise, within for three days on end, gather the sample of top and prematurity fringe with timed interval of 4 hours.Carry out the RNA atlas analysis with Agilent (Agilent) the corn array that is designed in about 105k probe, to detect the customization of full gene expression pattern.Being used for hundred million sensible (Illumina) digital gene, to express the sample of (DGE) platform be to collect 3 from 3 plant in per 4 hours to repeat samples within 1 day time.Then 3 samples being divided into 3 groups analyzes.
The GeneTS method is applied to data to determine periodically (Wichert, et al., (2004) Bioinformatics 20:5-20 (people such as Wichert,, " information biology ", the 20th volume, 5-20 page or leaf in 2004)).This method at first produces the periodogram of fourier frequency.Then assess the significance of remarkable fourier frequency with Fisher g statistical technique.In this given experimental design situation, this method has shown in detecting diel rhythm better renders a service (Hughes than other common methods, et al., (2007) the Cold Spring Harb Symp Quant Biol 72:381-386 (people such as Hughes, 2007, " cold spring port quantitative biology discussion procceedings ", the 72nd volume, 381-386 page or leaf); Hughes, et al., (2009) PLoS Genet 5:e1000442 (people such as Hughes,, " PLoS genetics ", the 5th volume, e1000442 page or leaf in 2009)).Then by being converted to the q value, relatively the significance value of checking from Fisher G is revised for multiple measurement, with estimation error discovery rate (Storey and Tibshirani, (2003) Proc Natl Acad Sci USA 100:9440-9445 (Storey and Tibshirani, 2003, " institute of NAS periodical ", the 100th volume, 9440-9445 page or leaf)).The transcript of regulation and control is confirmed as having at least once and significantly expresses and lead the transcript that also has significance when being 10% at FDR every day round the clock.
The round the clock microarray analysis of leaf
The diel rhythm of genetic expression can detect in photosynthesis leaf texture easily.In 44,187 probes with the expression that can detect, 10,037 or 22.7% are accredited as circulation with the GeneTS algorithm.(Hazen, et al., (2009) Genome Biol 10:R17 (people such as Hazen,, " genome biology ", the 10th volume, R17 page or leaf in 2009)) is consistent for the ratio of reporting in this ratio of cyclical transcription thing and the Arabidopis thaliana.Significantly the median cycle of the transcript of circulation is 24.1 hours, as desired under the natural condition.The amplitude of cyclical transcription thing is strong, and the median of peak/paddy ratio is about 5 times, the many peak that is higher than 20 times/paddy ratios that demonstrate.The peak value of these cyclical transcription things shows a very wide distribution, and the peak appears at all stages of one day.
The round the clock microarray analysis of fringe
Opposite with the result of leaf, in the growth period fringe, only have few transcript to show diel rhythm.In the transcript probe of 38,445 expression, only have 149 (1.7%) clearly to be accredited as to circulate.Although cyclical transcription thing quantity is few, but still have at dusk enrichment, the transcript of only about half of circulation peak value occurs in this stage.In above-mentioned 149 transcripts, 100 (67.1%) is diurnal cycle in leaf texture also.In the transcript that all circulates in leaf and fringe tissue, the amplitude of the rhythm and pace of moving things in the growth period fringe seriously weakens.Behind the note that merges unnecessary probe and more thorough gene, this inventory has reduced to 45 fringe cyclic genes (Fig. 3) of inferring.The corn homologous gene of many Arabidopis thaliana vibrator CCA1/LHY that seemingly describe in detail, TOC1, PRR7/3, GI, ZTL (ZEITLUPE is called Adagio sample protein 3 in paddy rice) in these genes.Mealie tissue core vibrator is therefore seemingly complete, but obviously and its great majority transcribe output system and removed coupling.
Yet some output genes have been found in the one group of gene that in fringe, circulates.The inventory of the transcript of strong circulation comprises that maximum 13 corns catch light CAB transcript (chlorophyll a-b conjugated protein), is the subset of larger corn C AB gene family.Be positioned No. 1 chromosomal CONSTANS sample (ZmCO sample) gene and circulate in fringe and leaf, it expresses peak value between the lights (6PM).Yet it is before to be accredited as to be positioned at chromosomal conz1 (Miller, et al., (2008) Planta 227:1377-1388 (people such as Miller No. 9,2008, " plant ", the 227th volume, 1377-1388 page or leaf)) different CO homologous genes.Detected the strong circulation of MYB sample transcription factor (ZmMyb.L), (6AM) reaches peak value at dawn.This gene is the homologous gene of REVEILLE1, and REVEILLE1 is the Myb transcription factor (people such as Rawat, (2009)) that in the Arabidopis thaliana circadian clock and growth hormone approach is combined.Two fringe specific genes have interesting estimation function, and zinc finger protein matter (ZmZF-5) reaches peak value at 10AM, and the serine/threonine protein matter kinases (ZmSAPK9) that osmotic stress/dormin activates reaches peak value at 6PM.In other cyclic genes, 3 coding translocators, 2 heat shock proteins, several enzymes and putative protein matter are arranged.
The digital gene expression analysis
Be that hundred million sensible (Illumina) DGE expresses platform (hundred million sensible limited-liability company specially, No. 9885, main road, center, town, the inferior San Diego of U.S. markon welfare, 92121 (Illumina, Inc., 9885 Towne Centre Drive, San Diego, CA 92121 USA) gathered independently sample, and analyzed rhythmicity.This has represented the first NexGen moldeed depth degree order-checking trial for determining that rhythmicity Circadian Expression pattern is carried out.To having carried out since the order-checking of the anchor point of two restriction enzyme site DPNII and NLAIII from 6 time points (ZT0, ZT4, ZT8, ZT12, ZT16 and ZT20), 3 parallel sample separately.The sample of each multiplication moves in independent wandering cells passage.Assessed the quality of output sequence and output sequence has been compared with respect to Dana Farber Gene Index Maize 19.0 (can the www.bio.dfci.harvard.edu/tgi/ on the World Wide Web find).Altogether 4.7 * 10 9Individual base pair has passed through all-mass control and the comparison index of order-checking process, and it is about each passage 1.3 * 10 8Bp.To surpassing 1.89 * 10 8Individual label carries out the gene expression analysis of rhythmicity behavior, namely about 5,250,000 labels of every duplicate samples.3 repetitions manually are divided into continuous 3 days.Then use the method identical with microarray data to assess the periodicity of data.These data are mainly used in confirming independently those cyclical transcription things of identifying by microarray strategy more strong on the statistics therefore being not used as herein the independent experiment of finding herein.
The result has shown with Agilent (Agilent) and has analyzed widely consistence.In leaf texture, 2559 transcripts are accredited as in leaf texture and circulate.Useful Agilent (Agilent) be accredited as that the core component of circulation also is confirmed as circulating under hundred million sensible (Illumina).Two kinds of technology all are accredited as 1378 transcripts that have of circulation.Because these transcripts are that each different atlas analysis platform is found independently, these transcripts are the highest base groups of degree of confidence of cyclical transcription thing in photosynthesis of maize (leaf) tissue.
Hundred million sensible (Illumina) atlas analysis of growth period fringe shows the cyclical transcription thing that surpasses 2 times of Agilent (Agilent), and wherein 362 demonstrate significant rhythmicity.Yet although the quantity of cyclic gene has increased in the growth period fringe, it is compared still seldom with leaf photosynthesis tissue.Although the consistence between these different technology is lower, 48 transcripts of circulation in two platforms, have still been identified from fringe.In the transcript of these 48 circulations, 23 are gone out by Agilent (Agilent) and hundred million sensible (Illumina) technical evaluation and all identify in leaf and fringe tissue.In remaining 25, in 3 times of 4 possible tests (leaf Agilent (Agilent), leaf hundred million sensible (Illumina), fringe Agilent (Agilent) and fringe hundred million sensible (Illumina)), there are 24 to be accredited as circulation.These independent results confirm that core oscillator works in the fringe tissue.
Circadian Expression is analyzed
Corn transcribe round the clock spectrum be strong and with Model Plants Arabidopis thaliana in independently biological tissue and technology platform to transcribe round the clock spectrum similar.There is diel rhythm from the result of the photosynthesis leaf texture that receives light for the transcript that goes out with Agilent (Agilent) technical evaluation up to the expression of 22.7% (10K/44K probe).Independently transcribe the class range analysis platform with two, be Agilent (Agilent) microarray and hundred million sensible (Illumina) label sequencing, compensate the intrinsic deviation of arbitrary technology, demonstrate 1400 round the clock lease core high confidence level groups of the transcript of regulation and control.
In non-photosynthetic growth period fringe, diel rhythm can not play remarkably influenced to transducer.Only having 45 genes only to be accredited as circulates in fringe or in fringe and leaf.13 CAB (Chlorophyll A/B transcript) have wherein been found, they are marker (the Millar and Kay of the Circadian Expression pattern of having established in the plant now, (1991) Plant Cell 3:541-550 (Millar and Kay, 1991, " vegetable cell ", the 3rd volume, the 541-550 page or leaf)).Yet, to compare with leaf, their amplitudes in fringe seriously weaken.11 ortholog genes of core oscillator system appear at this and stride and organize in leaf-fringe group.Therefore, as if core oscillator has activity in fringe.The core oscillator of plant is described to chain 3 or 4 loop procedure (Harmer, (2009); Ueda, (2006) Mol Syst Biol 2:60 (Ueda,, " molecular system biology ", the 2nd volume, the 60th page in 2006)).This presentation of results is by ZmCCA1/ZmLHY and ZmTOC1a, and the center feedback control loop that b forms is guarded in corn.This loop shows the ripple of extreme amplitude in leaf texture, may be as the main driving mechanism of transcribing output.In the fringe tissue, these wave amplitudes reduce, and reduce respectively 83% and 94%, main reducing by the peak value transcriptional level.The height that fringe organizes waveform to reduce strongly shows lasting diurnal cycle but amplitude reduces.As if it is not desynchronizing of pattern round the clock, and the latter may scatter skew in circulation pattern, and peak-paddy waveform is weakened or fog.If the ZmCCA1/ZmTOC1 loop is really as the center zeitgeber, because its waveform that weakens, it can seriously reduce the Relative Contribution of exporting round the clock the gene transmitted signal.For example comprise also significantly reducing of disclosing solution amplitude of ZmPRR73/ZmPRR37, gigz1/gigz2 and isogenic two external loop-arounds of ZmZTLa/ZmZTLb.
An explanation of core engine device in the fringe and output pathway uncoupling is attributable to low light intensity penetrates the growth period fringe by the shell leaf (bract) that wraps up fringe.The Circadian Expression pattern transcribe enhancing may by photosensitive protein for example phytochrome and cryptochrome occur, so this enhancing may correspondingly reduce in the fringe that relatively lacks light.As shown in the Arabidopis thaliana, core oscillator clock gene such as CCA1 and LHY are by the activation of the CAB gene of photoactivation and mediation output people such as (, (1997)) Wang.Therefore, the short arc of core oscillator may not produce enough protein and causes transcribing of output pathway or only can faintly cause.There is minority output gene (its promotor may be responsive to the core oscillator product of lower level) to be activated, effectively removed coupling but totally transcribe output.
The gene that only has a small amount of circulation in fringe can be the near-end translation node that connects core oscillator and output pathway.One of them is ZmMyb.L, and its expression peak in leaf and fringe is at 6AM.The height identity of the MYB structural domain of the gene C CA/LHY of the demonstration of ZmMYB.L protein and Arabidopis thaliana and corn in the stage in the morning, even expand to the SHAQKYFF protein motif that comprises uniqueness.ZmMyb.L may have the ortholog gene function of Arabidopis thaliana REVEILLE1, and REVEILLE1 is in conjunction with circadian clock and growth hormone approach (Rawat, (2009)).
The Arabidopis thaliana root that grows up to and the microarray analysis of seedling tissue show that the core oscillator that reduced form is arranged is at circulation (James in the non-photosynthesis tissue of root, et al., (2008) the Science 322:1832-1835 (people such as James, 2008, " science ", the 322nd volume, the 1832-1835 page or leaf)).According to above-mentioned microarray expression study, in the seedling tissue, there are 6518 transcripts to be accredited as circulation, by comparison, in root tissue, there are 335.These results conform to substantially with discovery disclosed herein; No matter that is to say, in the non-photosynthesis tissue of major part, be root or fringe, the many components operation of core oscillator, but it is transcribed output and greatly weakens.
Physiologic function round the clock
Studied round the clock the genetic expression rhythm and pace of moving things understanding better the round the clock biological scope of regulation and control at molecular level, and then the crop performance of can having an opportunity to improve.(Fig. 5) these results have disclosed round the clock many aspects of mechanism of corn, are delivered to downstream effect device gene from core clock gene, signal.The round the clock vibration of leaf of Semen Maydis genetic expression is general, and the circulation in rising and falling round the clock of thousands of genes and accompaniment functions thereof is arranged.The physiological role that obviously develops in one day scope is interesting, and has specifically shown the control by stages of expressing, but this also can be that near-end in the diel rhythm and far-end event are responded and the natural evolution of the physiological event that shows.Have recognized that, adopt thinner temporal resolution just can obtain more round the clock transcripts of regulation and control, can describe better simultaneously the differentiation that function in a day manifests degree.The round the clock atlas analysis investigation of this full genome is first for the research of corn, in conjunction with the distribution that surpasses 1700 function items, has disclosed in one day the continuously lasting profile of differentiation of function event.Be clear that diel rhythm is complicated, and interweave dearly in all biological process of cell.This is suitable for the expression that cell mechanism is consistent or coordinate by inference.
Existence at the bimodal function Enrichment Mode in the morning and afternoon/evening is interesting, almost reflects for certain a kind of basic activity in the daily work and rest of plant.More gene peak value occurs at the time point of 10AM and 6PM, and this is own so that the more functional classification under these genes also peak value occurs in these times, thereby forms this bimodal functional mode.Although each gene of regulating and control round the clock only the time in one day peak value appears, functional classification is that the bimodal fact means that the different genes under these functional classifications peak value occurs in the different time.It is also conceivable that now the possible relation (Yeang with " solarium " that be calibrated to noon of nearest description, (2009) Bioessays 31:1211-1218 (Yeang,, " biological short commentary " in 2009, the 31st volume, the 1211-1218 page or leaf)).What the peak in these mornings and evening can represent to carry out between the gene of the gene regulated and control round the clock and solarium regulation and control communicates by letter.
Pattern is very strong in leaf round the clock, but in the growth period fringe a little less than.The growth period fringe also is to experience the round the clock photosynthesis source organ's of the complications of vibration master library.Do not drive round the clock even immature fringe self does not have obvious inside, can expect to occur to receive round the clock driving (for example passing through ripple and the fixing carbon of movable signal) from the source organ, with the round the clock Transcription from some gene of external drive.But this does not obviously observe.The gene of regulating and control round the clock with regard to minority fringe in a day occurred with regard to time of peak value, function enrichment explanation signal transduction and transcribing in the morning, photosynthesis in the afternoon, core oscillator and transcriptional control are at night.
Core clock mechanism and come from this component of adjacent signal mechanism for example for example leaf and fringe Relations Among reach forward and affect the such mode of crop performance and improve change or extended source and storehouse.Round the clock pattern and the apparent adaptability (Ni, (2009)) of combination have been confirmed to strengthen from the extensive genetic complementation of the round the clock pattern in different germplasms source.
The genome structure of example 2.ZmCCA1 and ZmLHY
Find that in the process of the corn gene model of inferring ZmCCA1 and ZmLHY this gene is respectively by the gene region of about 45kb and 78kb encode (Fig. 4).The corn gene of this size is very rare, and its average gene size is near 4kb (Bruggmann, et al., (2006) Genome Res 16:1241-1251 (people such as Bruggmann,, " genome research " in 2006, the 16th volume, the 1241-1251 page or leaf)).The exon of ZmCCA1 and ZmLHY gene-intron model is to be derived by its cDNA sequence that obtains from the BAC order-checking and the comparison of genome sequence.The ZmCCA1 gene is comprised of 11 exons, and these exons by the intron of 10 different lengthss separately.The longest is intron #2 (about 9kb) and intron #6 (about 15.6kb), very rare in Maize genome.Translation initiation codon ATG is positioned at exon #5.This means that untranslated 5 ' UTR is divided into 5 the little exons of size in the 40-200bp scope.The ZmLHY gene is comprised of 10 exons, and these exons by the intron of 9 different lengthss separately.(may lose at the medium and small exon of existing EST).Intron 2 is about 30.0kb, and intron 6 is about 20.1kb, and they may be introns maximum in the Maize genome.Known control Regulation of Gene expression sequence is usually located at intron.Very long intron may work in ZmCCA1 and ZmLHY regulation and control.ZmCCA1 and ZmLHY gene are all extremely long.The gene of growing especially may slow down and transcribe, thereby is a kind of genome regulation and control form of genetic expression.Similar with ZmCCA1, translation initiation codon ATG is positioned at exon 5.The maturation of the complicated exons structure explanation Pre-mRNA of 5 ' UTR may be the regulation and control of these another levels of gene.
Dna sequencing
With the two strands Shutgun method (people such as Bodenteich at random, Shotgun cloning or the strategy of choice to generate template for high-throughput dideoxynucleotide sequencing (in order to generate Shutgun cloning or the selection strategy of high-throughput dideoxy nucleotide sequencing template), be stated from: M.D.Adams, C.Fields, J.C.Venter (Eds.), Automated DNA Sequencing and Analysis, Academic Press, San Diego, 1994, pp.42-50 (M.D.Adams, C.Fields, J.C.Venter edits, " automated DNA order-checking and analysis ", academic press, San Diego, 1994, the 42-50 page or leaf)) to the BAC cloning and sequencing.In brief, remove lysate scheme (double-acetate cleared lysate protocol) separation BAC clone by diacetate after, make its fracture and repair the gained fragment is terminal by atomizing, be subcloned among the pBluescript II SK (+).After being transformed into the DH-10B electricity by electroporation and turning competence intestinal bacteria (Escherichia coli) cell (hero (Invitrogen)), select instrument (Genetix) with automatic Q-Bot bacterium colony and choose bacterium colony and be placed in the freezing substratum that contains 6% glycerine and 100 μ g/ml penbritins and be stored in-80 ℃.Then use Templiphi dna sequencing template amplification kit method (General Electric's Medical Group (GEHealthcare)) separation quality grain.In brief, the Templiphi method is used phage
Figure BDA00002091896500861
Archaeal dna polymerase is by isothermal rolling-circle TRAP amplification cyclic single strand or double-stranded DNA (Reagin, et al., (2003) J.Biomol.Techniques 14:143-148 (people such as Reagin,, " biomolecules technology " in 2003, the 14th volume, the 143-148 page or leaf)).Then with amplified production at 95 ℃ of sex change 10min and in 384 orifice plates, carry out end sequencing, use carrier to cause M13 oligonucleotide and the ABIBigDye version 3.1 Prism sequencing kits of type during order-checking.After the removing based on ethanol, the cycle sequencing reaction product is heavy molten and detect at Perkin-Elmer ABI 3730xl automatic sequencer, and assemble each sequence with the Phred/Phrap/Consed software package (www.phrap.org/phredphrapconsed.html on the World Wide Web) of PD.Check and confirm contig order with Exgap (A.Hua, University of Oklahoma (University of Oklahoma), personal communications).Exgap is local graphical tool, and it uses the base pair reading information to the contig ordering of Phred, Phrap and Consed generation, and confirms the accuracy based on the assembling of Phrap.Subsequently, order-checking breach between most of contig of paying close attention to is closed in the following way: in the presence of the sequencing primer of custom design, previous plasmid DNA template with the amplification of Templiphi amplifing reagent cassette method is checked order, and the custom sequence of gained is inserted in the original assembling based on Phrap.Also by to checking order the breach sequence between the contig of paying close attention to of confirming to be left with the overlap of disclosed BAC dna sequence dna ZMMBBc0099K11 (GenBank AC211312.1) and the ZMMBBc0076L18 (GenBank AC213378.3) of American National biotechnology information center RiboaptDB (namely from).
The promotor that example 3. is regulated and control round the clock
Round the clock (daytime) circulation of light and temperature is the environmental factors that all living organisms adapt to.For example grow in nearly all aspect of plant physiology, growth, photosynthesis and photoassimilate partitioning, respiration, stress reaction, hormone response, nitrogen assimilation are all regulated and control round the clock.
The promotor of one day specified time provide according to naturally round the clock pattern handle in a controlled manner the instrument of specific physiology or metabolic process.For example, can cause participating in the rise of the gene of photosynthesis and carbohydrate metabolism in one day to the artificial downward modulation of daystart clock gene CCA1 and LHY, thereby improve growing way and output.For realizing downward modulation, CCA1 and LHY promotor can drive the RNAi expression cassette of himself.
Complete genomic round the clock RNA atlas analysis provides the material standed for high inducibility and low background for the promotor in each stage in one day.The root a tree name needs, and the example of one day concrete time is (for example transcribe 12h and open, 12h closes) of pulsation (be every day only of short duration transcribing once), broad peak or any position between the two.
The gene that relates to multiple Agronomic character (for example frost resistance, winter hardiness, drought tolerance, increase by improving the output that metabolism causes) is suitable for round the clock controlling element adjusting disclosed herein.Randomly, these element and tissue-specific promoter unite and use to optimize the required expression pattern of the gene of being paid close attention to round the clock.For example, in one embodiment, improve the gene of drought tolerance and under the co-controlling of the round the clock controlling element that shows the peak expression pattern about noon or dusk and root-specific promoter element, express.Similarly, the gene that improves winter hardiness and frost resistance is expressed under the co-controlling of the round the clock controlling element of the peak expression pattern that shows dawn or evening and leaf specificity promoter element.In addition, relating to during carbohydrate metabolism and the photosynthesis gene of source/base relation expresses under the co-controlling of round the clock promoter element disclosed herein and one or more tissue-specific promoters element.Known several genes relates to abiotic stress patience and nitrogen use efficiency (referring to for example U.S. Patent Application Publication No.US 2010/0223695; US 2010/0313304; US 2010/0269218).As shown in Figure 5, the gene that belongs to a plurality of functional classifications shows different Circadian Expression patterns.For example, GO:0009651 occurs peak value to the response of salt stress in the middle of the morning, and peak value appears in GO:0008643 carbohydrate transhipment at night.
From relational approach and the genes regulated and control altogether of those genes round the clock regulation and control also within the scope of the invention.These relational approaches member's expression is controlled with by using one or more round the clock controlling element disclosed herein to regulate and control better.
Promotor motif analysis method
Proved in the document that the combination of a small amount of motif is by length and destructive interference can be created in the waveform that any phase shift is issued to peak value mutually.(CBE:Wang for example, et al., (1997) Plant Cell 9:491-507 (people such as Wang, 1997, " vegetable cell ", the 9th volume, 491-507 page or leaf) and EE:Alabad í, et al., (2001) Science 293:880-883 (people such as Alabad í, calendar year 2001, " science ", the 293rd volume, the 880-883 page or leaf).Yet the degree of the quantity of these controlling elementss and its conservative property between plant species is not fully set forth.The promotor of described 144 corn genes is according to zeitgeber time (being the opportunity that its peak value is expressed) classification, wherein ZT0=6am, ZT4=10am, ZT8=2pm, ZT12=6pm, ZT16=10pm and ZT20=2am.To every group of promoter Analysis the existence of the motif identified in the edge species Arabidopis thaliana far away.Motif is " CBE ", " EE ", " O-G-box ", " Morning Element ", " SORLIP1 ", " Refined Morning Consesnus ", " Evening GATA ", " Telo Box ", " Starch Box " and " Protein Box ".These motifs are identified by literature search, comprise being accredited as the motif of expressing at the morning, evening and night.In the 2000bp of TSS with forward and reverse scan promotor, to obtain the exact matching of motif.
Table 2
Element Sequence SEQ ID
CBE AAAAATCT SEQ ID NO：472
CBE’ AGATTTTT SEQ ID NO：473
EE AAATATCT SEQ ID NO：474
EE’ AGATATTT SEQ ID NO：475
O G-Box GCCACGTG SEQ ID NO：476
O B-Box’ CACGTGGC SEQ ID NO：477
Morning Element AACCAC SEQ ID NO：478
Morning Element’ GTGGTT SEQ ID NO：479
SORLIP1 GCCAC SEQ ID NO：480
SORLIP1’ GTGGC SEQ ID NO：481
Refined Morning Element CCACAC SEQ ID NO：482
Refined Morning Element’ GTGTGG SEQ ID NO：483
Evening GATA GGATAAG SEQ ID NO：484
Evening GATA’ CTTATCC SEQ ID NO：485
TeloBox AAACCCT SEQ ID NO：486
TeloBox’ AGGGTTT SEQ ID NO：487
StarchBox AAAGCCC SEQ ID NO：488
StarchBox’ GGGCTTT SEQ ID NO：489
Protein Box ATGGGCC SEQ ID NO：490
Protein Box’ GGCCCAT SEQ ID NO：491
From widely literature search, collected the diel rhythm motif, having comprised:
CBE:Carr é and Kay, (1995) Plant Cell 7 2039-2051 (Carr é and Kay, nineteen ninety-five, " vegetable cell ", the 7th volume, 2039-2051 page or leaf).EE:Harmer and Kay, (2005) Plant Cell 17 1926-1940 (Harmer and Kay,, " vegetable cell ", the 17th volume, 1926-1940 page or leaf in 2005).
G-BOX, TELO, STARCH, PROTEIN and GATA:Michael, et al., (2008) .PLoS Genet.4e14 (people such as Michael,, " PLoS genetics ", the 4th volume, e14 page or leaf in 2008).SORLIP and Refined Morning Consensus:Hudson and Quail, (2003) Plant Physiol.1331605-1616 (Hudson and Quail,, " plant physiology ", the 133rd volume, 1605-1616 page or leaf in 2003).Morning Element:Harmer and Kay, (2005) Plant Cell 17 1926-1940 (Harmer and Kay,, " vegetable cell ", the 17th volume, 1926-1940 page or leaf in 2005).
EE and CBE motif from some genes have been made up hidden Markov model (HMM), described gene comprise remarkable circulation and with the motif of its Arabidopis thaliana ortholog gene in same suitable step cycle.These HMM do not have to show the preference to base around any, therefore use accurate core motif allow further to analyze.From the sequence of place, find out the exact matching of motif and reverse complemental.The gene dosage found and motif sum are compared to seek enrichment with respect to random chance with respect to the remainder of this group.
The motif analysis result
" CBE motif " is a 8bp motif, is called again the CCA1 binding member, and it should occur 13 times in the group of this analysis scale at random; In 144 promotors, the CBE motif has occurred 40 times accurately.The CBE motif is enriched in the gene that the time-division is found by day, and it follows the expression pattern (comprising in the present invention) of the corn ortholog gene of Arabidopis thaliana CCA1.
" EE motif " is a 8bp motif, is called again Evening Element, and it should occur 13 times in the group of this analysis scale at random; In 144 promotors, the EE motif has occurred 34 times accurately.In addition, generally the occurring of this motif concentrate on corresponding evening and night the peak value gene those promotors, this motif of＞40% is positioned at the promotor of ZT12 group,＞70% situation is between the 6pm-2am.In those genes that expressing at the peak appears in ZT12, there are 12 to comprise at least 1 EE in 23 genes.
" O-G-Box " is accredited as and drives motif the morning, and the data of this paper show in the O-G-Box motif of whole discoveries 50% first time point ZT4 after being positioned at illumination and beginning.Other elements in morning " Morning Element ", " SORLIP1 " and " Refined Morning Element " show similar pattern, the peak is enriched in illumination and begins rear back to back those time points (being respectively 28%, 33% and 31%), is that the theory of optical drive conforms to these promotors.What also conform to therewith is such fact: if cultivate plants to produce primary data by the long time on daytime, then the existence of these promotors is selected for putting ZT16 and ZT20 two real interlunations.
" Evening GATA ", " Telo Box ", " Starch Box " and " Protein Box " motif all be accredited as evening and the late into the night motif.At this, being defined as the time point at noon, when illumination is the brightest, there is the low enrichment of these motifs.These elements are relatively wide distribution range and the different peaks of a plurality of motif combination results expression phase unanimously theoretical with the time point at night in whole evenings.
Must notice that many in 144 promotors that identify are carried a more than motif, the median of the motif of finding in each promotor is 4, and the maximum motif number of discovery is 12.12 promotors wherein do not comprise any motif on each time point.In ZT12 peak value group (comprising highly general EE motif), there are 11 not comprise typical EE in 23 genes, and as previously mentioned, several genes do not comprise any known motif, this shows other factors and motif in action, so that observe the waveform of high amplitude, yet they also can be contained in the promoter sequence disclosed herein.
Promoter expression is analyzed
The seedling preparation
As follows with GS3 seed sterilization and make it to prepare to sprout: with 70% washing with alcohol 5 minutes, then with containing 50% SYNTHETIC OPTICAL WHITNER and 2 20 solution washing 15 minutes.Then with sterilized water washing 3 times, be respectively 5,15 and 5 minutes.Washed seed 5 minutes with 30% hydrogen peroxide, then with sterilized water washing 3 times.Then seed is placed in the sterilized water and soaked 5 hours.
With the moistening aseptic warm nursery paper of 15ml sterilized water and place aseptic Q pallet.Put into 16 seeds with the interval that equates at each pallet, cover with another aseptic warm nursery paper, and drench with the 9ml sterilized water.With Austraseal rubber belt sealing Q pallet, and place 22 ℃ of illumination cultivation chambers, cultivated 3 days.
Remove the kind leather material of parcel growth period seedling, and the seedling that sprouts is seeded on the substratum (pH 5.6) that contains 4.3%MS basis salt, 0.1% inositol, 0.5%MS VITAMIN liquid storage and 40% sucrose each plating 2 strain.
The preparation of leaf and bombardment
From 2 1/ 2 to 3 weeks large GS3 seedling tender leaf (part is exposed) 1 inch wide transverse section of separation, and it is upper in order to carry out bombardment to be seeded in the substratum (pH 5.6) that contains 4.3%MS basis salt, 0.1% inositol, 0.5%MS VITAMIN liquid storage and 40% sucrose.
The embryo preparation
With the prematurity maize of the plasmid bombardment that contains the gus gene that effectively is connected with test starting from greenhouse donor plant.The following conversion.
Collect corn GS3 fringe after 8-14 days and 30% in pollination
Figure BDA00002091896500922
Surface sterilization is 20 minutes in SYNTHETIC OPTICAL WHITNER and the 0.5%Micro washing composition, then uses aseptic water washing 2 times.Downcut immature embryo and with the plumular axis side down the direction of (the scultellum side up) place each dull and stereotyped 25 embryo.With these embryos in front 4 days dark culturing in the 560L substratum of bombardment.The 560L substratum is based on the substratum of N6, comprises Eriksson VITAMIN, VitB1, sucrose, 2,4-D and Silver Nitrate.On bombardment same day, embryo is transferred in the 560Y substratum 4 hours, then be arranged in the target region of 2.5cm.The 560Y substratum is that height oozes substratum (the 560L substratum that contains high concentration sucrose).After bombardment, embryo is maintained at 560Y substratum (a kind of substratum based on N6) upper 1 day, then dyeing is observed GUS and is expressed.
Bombardment
Prepare as follows DNA/ gold grain mixture and be used for bombardment: wash in advance the gold grain of 60mg0.6-1.0 micron with ethanol, with aseptic distillation H 2The O flushing also is resuspended in the altogether aseptic H of 1mL 2Among the O.The 0.6 μ M gold grain that washs in advance by supersound process 25 μ L, and join in the test plasmid of 20 μ L100ng/ μ L, DNA is deposited in the gold grain surface.With this mixture supersound process and add 2.5 μ L TFX again.This solution is placed upper 10 minute of vortex oscillation device of low speed setting.Then with this solution centrifugal 1min under 10K RPM, and from pipe, remove liquid.Add 60 μ L ethanol, then with this solution supersound process again.Then before bombardment, place this DNA/ gold mixture of 10 μ L on each huge carrier and make it dry.
Under the vacuum of the 27-28 inch of mercury, leaf and seedling are organized in 1100psi, embryo is bombarded seedling with the PDS-1000/He rifle under 450psi.Huge carrier and stop distance between the screen be 6 and 8cm between.After the bombardment flat board is cultivated 18-24h at 27-28 ℃ in sealed vessel.2 flat boards of each construct are cultivated in the dark, and 2 flat boards are cultivated under illumination in addition.
By being immersed, seedling contains 100mM NaH 2PO 4-H 2O (pH 7.0), 10mM EDTA, 0.5mM K 4Fe (CN) 6-3H 2In the GUS analysis buffer of O, 0.1%Triton X-100 and 2mM 5-bromo-4-chloro-3-indyl glucosiduronate, analyze instantaneous GUS expression in the tissue after bombarding.With this tissue 37 ℃ of incubation 24h in the dark.Change GUS dyeing solution termination analysis with 70% ethanol.Examine under a microscope GUS and express/dyeing.
BMS transforms
Containing 40ml#237 substratum (4.3%MS basis salt, 0.1% inositol, 0.5%MS VITAMIN liquid storage, 0.002%2,4-D and 40% sucrose, pH 5.6) the 250ml Erlenmeyer flask in, in the dark, shook lower cultivation BMS (Mexico's black sweet corn) cell 3 days with about 150RPM under 28 ℃.At this moment, add the 25ml#237 liquid nutrient medium and make again continued growth 3 days of culture, Agrobacterium-mediated Transformation may occur in this moment.At 1 day before this, the Agrobacterium culture that will contain the plasmid that comprises the gus gene that effectively is connected with test starting placed and contains suitable antibiotic 10ml culture, and makes it 28 ℃ of grow overnight.
Place laminar flow hood 10 minutes to allow cell settlement each 250mL Erlenmeyer flask.Remove the 20ml supernatant liquor.Move on to remaining mixture in the 50ml pipe and at the centrifugal 5min of 3200RPM.Remove supernatant liquor and use 40ml 561Q changing liquid cultivation matrix.561Q is based on the substratum of 4%N6, comprises Eriksson VITAMIN (1X), 0.005% VitB1,68.5% sucrose, 0.0015%2, and 4-D, 0.69%L-proline(Pro) and 36% glucose, pH are 5.2.With cell again at the centrifugal 5min of 3200RPM.Cell is resuspended in the final volume that reaches 15ml among the 561Q, and is divided into the 7.5ml aliquots containig in the 125ml Erlenmeyer flask.
Then with Agrobacterium culture at 3200RPM centrifugal 5 minutes, remove supernatant liquor, precipitation is resuspended in the 2ML 561Q+ Syringylethanone (AS).Syringylethanone solution is by preparation 100mM solution preparation in DMSO.This solution is added among the 561Q by 1 μ L A.S./1mL#561Q.Measure the cell concn of absorbancy to be identified for transforming of OD550.Be 0.75 o'clock at OD550,1ml Agrobacterium solution is added among the 5ml 561Q+AS, then shake common cultivation 3 hours with the speed of 150RPM under 28 ℃ in the dark with 7.5ml BMS cell.
Behind 3 hours incubations, add more 561Q substratum so that its volume reaches about 48ml in the 50ml pipe to the culture of this 13.5ml.The 12ml culture is coated on the aseptic filter paper dish, then be placed on the flat board of 562U substratum, in 28 ℃ of dark, cultivated 4 days.562U is based on the substratum of 4%N6, comprises Eriksson VITAMIN (1X), 0.005% VitB1,30% sucrose, 0.002%2, and 4-D, 0.69%L-proline(Pro), pH are 5.8.Then filter paper is moved on on the 563N flat board, in 28 ℃ of dark, cultivated again 2 days.563N is based on the substratum of 4%N6, comprises Eriksson VITAMIN (1X), 0.005% VitB1,30% sucrose, 0.0015%2, and 4-D, 0.69%L-proline(Pro) and 0.5%MES damping fluid, pH are 5.8.
For each test builds body is set up 4 flat boards.Two BMS flat boards of each construct are taken out and to GUS dyeing from dark, and other two then in that dyeing is prepended under the illumination 5 hours to GUS.The BMS cell scraped from filter paper put into new pipe and comprise 100mMNaH by cell is immersed 2PO 4-H 2O (pH 7.0), 10mM EDTA, 0.5mM K 4Fe (CN) 6-3H 2The GUS analysis buffer of O, 0.1%Triton X-100 and 2mM 5-bromo-4-chloro-3-indyl glucosiduronate is carried out instantaneous GUS expression analysis.With this tissue 37 ℃ of incubation 24h in the dark.Change GUS dyeing solution termination analysis with 70% ethanol.Examine under a microscope GUS and express/dyeing.
Representative promoter expression result
Zm-SARK PRO(PCO646468)
When adopting ZM-SARK PRO construct, in the bombardment of sprouting seedling, detected expression, but in leaf or embryo bombardment or BMS conversion, do not detected.
Zm-CCA PRO(PCO651594)
When adopting ZM-CCA PRO construct, in every kind of types of organization of test, all detected expression.
ZM-LHY PRO：ADH1 INTRON(PCO639678)
When adopting ZM-LHY PRO construct, in the bombardment of embryo, detected expression, but in leaf or seedling bombardment or BMS conversion, do not detected.
ZM-LHY PRO(ALT1)(PCO639678)
When adopting ZM-LHY PRO (ALT1) construct, all detecting expression in the bombardment experiment, but in BMS transforms, do not detecting.
ZM-NIGHT2 PRO(PCO643174)
When adopting ZM-NIGHT2 PRO construct, in the bombardment of embryo and leaf, detected expression, but in embryo bombardment or BMS conversion, do not detected.
ZM-NIGHT1 PRO(PCO503721)
When adopting ZM-NIGHT1 PRO construct, in the tissue of test, do not find detectable expression.Also might be under used test condition, to fail to capture the Circadian Expression pattern.
ZM-LICH2 PRO(PCO642613)
When adopting ZM-LICH2 PRO construct, in every kind of tissue of test, all detected expression.
Conversion and the regeneration of example 4. transgenic plant
With the prematurity maize of plasmid bombardment from the greenhouse donor plant, described plasmid contains can effectively be connected to drought-inducible promoter RAB17 promotor (Vilardell, et al., (1990) the Plant Mol Biol 14:423-432 (people such as Vilardell, nineteen ninety, " molecular biology of plants ", the 14th volume, 423-432 page or leaf)) transforming sequence and give selected marker PAT for the resistance of weedicide bialaphos.Perhaps, this selected marker provides at independent plasmid.The following conversion.Culture medium prescription sees below.
The preparation of target tissue:
Fringe shelled and 30%
Figure BDA00002091896500951
SYNTHETIC OPTICAL WHITNER added in the 0.5%Micro washing composition surface sterilization 20 minutes, then used twice of rinsed with sterile water.Immature embryo is downcut, and with (the scultellum one side up) placement down of plumular axis one side, 25 embryos of every plate were placed 4 hours at the 560Y substratum, then were in line to prepare to bombard in the 2.5cm target area.
The preparation of DNA:
The preparation plasmid vector, this plasmid vector comprises the transforming sequence that can effectively be connected to ubiquitin promoter.Use following CaCl 2The precipitation program adds that with this plasmid DNA the plasmid DNA that contains the PAT selected marker is deposited on the tungsten bead of 1.1 μ m (mean diameter):
The sub-aqueous solution of tungsten particle of 100 μ l preparation
(1 μ g) DNA (the total DNA of 1 μ g) in the 10 μ l Tris edta buffer liquid
100μl 2.5M CaCl 2
10 μ l 0.1M spermidines
Every kind of reagent sequentially is added to the sub-suspension of tungsten particle, remains on simultaneously on the multiple tube scroll machine.Final mixture is carried out of short duration supersound process, be allowed to condition at constant whirlpool and mixed lower incubation 10 minutes.At the precipitation after date, each pipe is carried out of short duration centrifugal, remove liquid, with 500ml 100% washing with alcohol, centrifugal 30 seconds.Again remove liquid, 105 μ l, 100% ethanol is added to the sub-bead of final tungsten particle.For particle gun bombardment, tungsten/dna particle is carried out of short duration supersound process, and get 10 μ l points and drip in the central authorities of each huge carrier (macrocarrier), allow its drying bombard after about 2 minutes.
Particle gun is processed:
Sample panel is bombarded with horizontal #4 in particle gun #HE34-1 or #HE34-2.All samples is accepted the single shooting of 650PSI, and the preparation particle/DNA of every pipe gets ten aliquots containigs altogether.
Subsequent disposal:
After bombardment, embryo is remained on 560Y substratum upper 2 day, then transfer to the 560R that contains the 3mg/L bialaphos and select substratum, divide training every 2 weeks.After the selection of carrying out about 10 week, the callus of anti-selection clone is transferred to the 288J substratum to cause plant regeneration.(2-4 week) transfers to well-developed somatic embryo the culturing room of germinateing in the substratum and transferring to illumination after the somatic embryo maturation.Approximately after 7-10 days, the plantlet of growing is transferred to 272V in the pipe without hormone culture-medium 7-10 days, until plantlet is grown fully.Then plant is transferred to the plate-inserting hole (being equivalent to 2.5 inches basins) that contains potting soil, 1 week of growth subsequently a regrowth 1-2 week in the greenhouse, is then transferred to typical 600 basins (1.6 gallons) and grows to maturation in the growth room.Plant is detected and mark for the drought tolerance that increases.The assay method of the drought tolerance of measure improving is the routine work of this area, for example comprise when with drought condition under the kernel under the same environmental conditions-heading ability output increase relatively time of contrast milpa.Select as another kind, can monitor transformed plant for the adjusting (being the reduction that small ear forms on the fringe) that meristematic tissue is grown.Referring to for example Bruce, et al., (2002) Journal of Experimental Botany 53:1-13 (people such as Bruce,, " experimental botany magazine ", the 53rd volume, 1-13 page or leaf in 2002).
Blast technique and substratum:
Bombardment substratum (560Y) comprises 4.0g/l N6 basis salt (SIGMA C-1416), 1.0ml Eriksson vitamine mixture (1000X SIGMA-1511), 0.5mg/l thiamine hydrochloride, 120.0g/l sucrose, 1.0mg/l 2,4-D and 288g/l L-PROLINE (transferring to the pH 5.8 rear deionized water constant volumes of using with KOH); 2.0g/l (after with the deionized water constant volume, adding) and 8.5mg/l Silver Nitrate (behind medium sterilization and cool to room temperature, adding).Select substratum (560R) to comprise 4.0g/l N6 basis salt (SIGMA C-1416), 1.0ml Eriksson vitamine mixture (1000X SIGMA-1511), 0.5mg/l thiamine hydrochloride, 30.0g/l sucrose and 2.0mg/l 2,4-D (transferring to the pH 5.8 rear deionized water constant volumes of using with KOH); 3.0g/l Two the third ammonia phosphorus (all behind medium sterilization and cool to room temperature, adding) of (after with the deionized water constant volume, adding) and 0.85mg/l Silver Nitrate and 3.0mg/l.
Plant regeneration substratum (288J) comprises 4.3g/l MS salt (GIBCO 11117-074), 5.0ml/l MS VITAMIN liquid storage (0.100g nicotinic acid, 0.02g/l thiamine hydrochloride, 0.10g/l pyridoxine hydrochloride and 0.40g/l glycine, with refining deionized water constant volume) (Murashige and Skoog, (1962) Physiol.Plant.15:473 (Murashige and Skoog, 1962, " plant physiology ", the 15th volume, the 473rd page)), the 100mg/l inositol, 0.5mg/l zeatin, 60g/l sucrose and 1.0ml/l 0.1mM dormin (it is rear with refining deionized water constant volume to transfer to pH 5.6); 3.0g/l
Figure BDA00002091896500973
Two the third ammonia phosphorus of (after with the deionized water constant volume, adding) and 1.0mg/l indolylacetic acid and 3.0mg/l (all adding at medium sterilization and after being cooled to 60 ℃).Comprise 4.3g/l MS salt (GIBCO 11117-074), 5.0ml/l MS VITAMIN mother liquor (0.100g/l nicotinic acid, 0.02g/l vitamin, 0.10g/l pyridoxine hydrochloride and 0.40g/l glycine are with refining deionized water constant volume), 0.1g/l inositol and 40.0g/l sucrose (pH 5.6 is rear with refining deionized water constant volume transferring to) and 6g/l bacto without hormone culture-medium (272V) TMAgar (after with refining deionized water constant volume, adding), aseptic and be cooled to 60 ℃.
The conversion that example 5. is agriculture bacillus mediated
For the antisense sequences with transforming sequence of the present invention corn is carried out agriculture bacillus mediated conversion, preferably adopt the method (U.S. Patent No. 5 of Zhao, 981,840 and the PCT patent announce No.WO98/32326, the content of described patent is incorporated this paper accordingly by reference into).Briefly, isolate immature embryo and embryo is contacted with the suspension of Agrobacterium from corn, wherein this bacterium can be transferred to transforming sequence at least one cell (step 1: infect step) of at least one immature embryo.In this step, preferably immature embryo is immersed in the agrobacterium suspension for beginning inoculation.Embryo and Agrobacterium are cultivated for some time altogether (step 2: be total to culturing step).Preferably, after infecting step, immature embryo is cultivated at solid medium.After this common incubation period, be susceptible to optional " tranquillization " step.In this tranquillization step, embryo is carried out incubation at least a known can the inhibition in the presence of Agrobacterium growth antibiotic, do not add the selective agent (step 3: the tranquillization step) of vegetable transformant.Preferably immature embryo is cultivated with microbiotic on solid medium, but indiscriminate dose, be used for eliminating Agrobacterium and for the quiescent stage of infected cell.Then, will cultivate at the substratum that contains selective agent through the embryo of inoculation, reclaim the transformed calli (step 4: select step) that grows.Preferably, immature embryo is cultivated with selective agent on solid medium, thereby caused the selective growth of transformant.Then be plant (step 5: regeneration step), and preferably will cultivate to regenerate plant at solid medium at the callus that selective medium is grown with callus regeneration.The adjusting of growing for meristematic tissue is monitored plant and is marked.For example, the size of seedling and floral meristem and the change of outward appearance and/or the output increase of leaf, flower and/or fruit are monitored.
The example 6. corns round the clock expression of crossing of gene affect plant size and growth
Express the round the clock function of gene of this genetically modified transgenic plant detection by using.Confirm transgene expression with transgenosis Auele Specific Primer RT-PCR.
Nourish and grow and biomass accumulation:
The close relative compares with non-transgenic, the increase that transgenic plant (T1 generation) should show plant height.By comparing the diameter stem value of transgenic plant and unconverted control group, measure the stem of transgenic plant.The increase of plant height and stem thickness can cause the larger plant height of transgenic plant and biomass.
Have been found that gene is mainly by accelerating growth velocity rather than prolong growth cycle to affect plant-growth round the clock.As if the growth that strengthens (the plant size and the biomass accumulation that namely increase) mainly is owing to the growth velocity of accelerating rather than owing to the growth cycle that prolongs, because according to weaving silk and bloom the date, the not delay of blooming of transgenic plant.Therefore, the growth velocity of plant is accelerated in the expression meeting of crossing of gene round the clock.As if the growth velocity of accelerating relevant with the round the clock speed that increases.
Increase in the transgenic plant nourish and grow, the growth velocity of biomass accumulation and quickening can use a large amount of field experiments to the advanced generation (T3) of hybridization and inbreeding background further to detect.The transgenic plant expection shows following one or more characteristics: plant height increases, diameter stem increases, the stalk dry weight increases, leaf area increases, the plant gross dry weight increases.
Reproductive growth and Grain Yield:
Round the clock gene cross to express may with strengthen the germinal tissue growth correlation.The expection of T1 transgenic plant can show following one or more characteristics: the seed gross weight increase of the increase of spike length degree, each fringe, seed quantity and the rice size of each fringe increase.The just variation of seed and fringe characteristic increases relevant with Grain Yield.
Reproductive growth and Grain Yield that transgenic plant have increased are confirmed a large amount of field experiment of advanced generation (T3).In inbreeding and hybrid context, all observed enhancing.Compare with non-transgenic close relative in contrast, the transgenic plant expection shows following one or more remarkable increase: main fringe dry weight, inferior fringe dry weight, fringe silk dry weight and shell dry weight.
Also assessed the stress tolerance parameter of transgenic plant, these parameters comprise: the abortion seed quantity of the bald point of the ASI of minimizing, minimizing and minimizing.When cultivating plants under high plant density stress conditions, the minimizing degree may be larger.The minimizing of these parameters is measured usually relevant with biological stress tolerance.
Example 7. is the variant of sequence round the clock
A. do not change the variant nucleotide sequence of the round the clock sequence of coded aminoacid sequence
With round the clock nucleotide sequence generation variant nucleotide sequence, the open reading frame nucleotide sequence that described variant nucleotide sequence has is compared with the initial unaltered ORF nucleotide sequence of corresponding SEQ ID NO has about 70%, 75%, 80%, 85%, 90% and 95% nucleotide sequence homology.These functional varianies produce with the standard cipher sublist.Although the nucleotide sequence of variant is changed, the coded aminoacid sequence of open reading frame does not change.
B. the variant aminoacid sequence of polypeptide round the clock
Produced the round the clock variant aminoacid sequence of polypeptide.In this example, change an amino acid.Particularly, observe open reading frame to determine suitable amino acid change.Select amino acid to change by considering protein comparison (with other ortholog genes or comparing from other gene families member of various species).Select such amino acid, it is considered to not be in (not high conservative) under the high selective pressure, and is quite easily had the amino-acid substitution of similar chemical property (being similar function side chain).Utilize the protein comparison, can suitable amino acid be changed.In case identify target amino acid, just carry out subsequently the program described in the following C part.Use this method to produce the variant with about 70%, 75%, 80%, 85%, 90% and 95% nucleotide sequence identity.
C. the extra variant aminoacid sequence of polypeptide round the clock
In this example, produce the artificial proteins sequence that for reference protein matter sequence, has 80%, 85%, 90% and 95% identity.This rear trial requires to differentiate conserved regions and variable region from comparison, then uses advisably the amino-acid substitution table.These parts will be described in a more detailed discussion.
Mainly, based on each round the clock between the protein or the conserved regions between other polypeptide make the decision which aminoacid sequence will change.Based on sequence alignment, the probably reformed regional of polypeptide represents with lowercase, and conserved regions represents with capitalization.It should be understood that and in following conservative region, to make conservative substitution and don't change function.In addition, the technician will appreciate that the functional variant of sequence of the present invention can have small non-conservative amino acid change in conserved domain.
Then produce with the urporotein sequence and differ artificial proteins sequence in 80-85%, 85-90%, 90-95% and 95-100% identity scope.Target fixes on the mid point of these scopes, and the positive and negative deviation approximate range for example is 1%.Amino-acid substitution will be realized by the perl script of customization.Permutation table provides in following table 3.
Table 3. permutation table
Amino acid Strong similar and displacement the best Change arranged sequentially Note
I LV
1 Displacement in 50: 50
L I，V 2 Displacement in 50: 50
V I，L 3 Displacement in 50: 50
A G 4
G A 5
D E 6
E D 7
W Y 8
Y W 9
S T 10
T S 11
K R 12
R K 13
N Q 14
Q N 15
F Y 16
M L 17 First methionine(Met) can not change
H Na Without good substitute
C Na Without good substitute
P Na Without good substitute
At first, identify any conserved amino acid that should not change in the protein also " mark on the work " do not do displacement to keep apart.Initial methionine will be added to this list certainly automatically.Then, make change.
H, C and P change in no instance.This change will at first begin with Isoleucine, is scanned up to the C end from the N end.Then be leucine, so by tabulating down until reach required target.Can make middle number displacement (interim number substitution), in order to do not cause the reverse of change.The order of list is 1-17, therefore changes beginning with Isoleucine as much as possible as required, then is leucine, until methionine(Met).Obviously, in this way, many amino acid will not need to change.L, I and V will be referred to displacements in 50: 50 of two optimal displacement that replace.
The variant aminoacid sequence is write out as output.Calculate identity percentage ratio with perl script.Use this program, produce the variant that has the polypeptide of about 80%, 85%, 90% and 95% amino acid identity with the initial unaltered ORF nucleotide sequence of following SEQ ID NO: 1,3,5 and 40-71.
Example 8. uses controlling element disclosed herein and polypeptide to change the proterties of plant
Various controlling element disclosed herein can be used for (comprising round the clock promotor and round the clock polypeptide) the proterties exploitation of various crop.This comprises engineered anti-freezing or nutritious compound absorption, source/storehouse adjusting, disease resistance, insect-resistant and the insect-resistance of photorespiration, the stomatal aperture regulation and control of frost resistance, cold-resistant or winter hardiness, drought-enduring or thermotolerance, salt stress patience, reduction, the nitrogen utilization that makes photosynthetic efficiency, carbohydrate metabolism and transhipment that output increases, increase, the biosynthesizing of selectivity metabolite, improvement.One or more controlling element disclosed herein and other controlling elements (comprising the derivable or tissue specificity motif of various abiotic stress) combination is to optimize transgene expression.
All publications in this specification sheets and applications for patents understand the common skill level in the affiliated field of the present invention.This paper is all incorporated in all publications and patent application by reference into, the degree of quoting just as each independent publication or patent application by particularly with to point out independently to incorporate by reference this paper into the same.
Through each concrete and preferred embodiment and technical description the present invention.However, it should be understood that, can make many variations and modification remaining under the prerequisite of the spirit and scope of the present invention.
Figure IDA00002091897100011
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Claims (38)

1. the polynucleotide of a separation, the polynucleotide of described separation are selected from:
A. polynucleotide, as determining under default parameters with the GAP algorithm, described polynucleotide be selected from SEQ ID NO:1,2,3,4,5,6,7,8,20,184,186,188,190,192,194,196,198,200,202,204,206,208,210,212,214,216,218,220,222,224,226,228,230,232,234,236,238,240,242,244,246,248,250,252,254,256,258,260,262,264,266,268,270,272,274,276,278,280,282,284,286,288,290,292,294,296,298,300,302,304,306,308,310,312,314,316,318,320,322,324,326,328,330,332,334,336,338,340,342,344,346,348,350,352,354,356,358,360,362,364,366,368,370,372,374,376,378,380,382,384,386,388,390,392,394,396,398,400,402,404,406,408,410,412,414,416,418,420,422,424,426,428,430,432,434,436,438,440,442,444,446,448,450,452,, 454,456,458,460,462,464,466, the full length sequence of 468 and 470 polynucleotide has at least 90% sequence identity; Wherein said polynucleotide encoding polypeptide, described polypeptide has the function of diel movement conditioning agent;
B. polynucleotide, described polynucleotide are selected from SEQ ID NO:1,2,3,4,5,6,7,8,20,184,186,188,190,192,194,196,198,200,202,204,206,208,210,212,214,216,218,220,222,224,226,228,230,232,234,236,238,240,242,244,246,248,250,252,254,256,258,260,262,264,266,268,270,272,274,276,278,280,282,284,286,288,290,292,294,296,298,300,302,304,306,308,310,312,314,316,318,320,322,324,326,328,330,332,334,336,338,340,342,344,346,348,350,352,354,356,358,360,362,364,366,368,370,372,374,376,378,380,382,384,386,388,390,392,394,396,398,400,402,404,406,408,410,412,414,416,418,420,422,424,426,428,430,432,434,436,438,440,442,444,446,448,450,452,, 454,456,458,460,462,464,466,468 and 470;
C. with the polynucleotide of (a) or polynucleotide complete complementary (b);
D. by the polypeptide of (a) or polynucleotide encoding (b); With
E. polypeptide, as determining under default parameters with the GAP algorithm, described polypeptide be selected from SEQ ID NO:185,187,189,191,193,195,197,199,201,203,205,207,209,211,213,215,217,219,221,223,225,227,229,231,233,235,237,239,241,243,245,247,249,251,253,255,257,259,261,263,265,267,269,271,273,275,277,279,281,283,285,287,289,291,293,295,297,299,301,303,305,307,309,311,313,315,317,319,321,323,325,327,329,331,333,335,357,359,361,363,365,367,369,371,373,375,377,379,381,383,385,387,389,391,393,395,397,399,401,403,405,407,409,411,413,415,417,419,421,423,425,427,429,431,433,435,437,439,441,443,445,447,449,451,453,455,457,459,461,463,465,467,467, the full length sequence of 469 and 471 polypeptide has at least 90% sequence identity.
2. recombinant expression cassettes, described recombinant expression cassettes comprises polynucleotide claimed in claim 1, and wherein said polynucleotide effectively are connected to promotor with the sense or antisense orientation.
3. host cell, described host cell comprises expression cassette claimed in claim 2.
4. transgenic plant, described transgenic plant comprise recombinant expression cassettes claimed in claim 2.
5. transgenic plant according to claim 4, wherein said plant is monocotyledons.
6. transgenic plant according to claim 4, wherein said plant is dicotyledons.
7. transgenic plant according to claim 4, wherein said plant is selected from: corn, soybean, Sunflower Receptacle, Chinese sorghum, canola oil dish, wheat, clover, cotton, paddy rice, barley, millet, peanut, sugarcane and cocoa.
8. transgenic seed, described transgenic seed is from transgenic plant claimed in claim 4.
9. modulate the circadian method of plant for one kind, described method comprises:
A. introduce recombinant expression cassettes in vegetable cell, described recombinant expression cassettes comprises the polynucleotide claimed in claim 1 that effectively are connected to promotor; With
B. under the plant cell growth condition, cultivate described plant; Described diel rhythm in the wherein said vegetable cell is modulated.
10. method according to claim 9, wherein said vegetable cell is from being selected from following plant: corn, soybean, Sunflower Receptacle, Chinese sorghum, canola oil dish, wheat, clover, cotton, paddy rice, barley, millet, peanut, sugarcane and cocoa.
11. whole strain plant or circadian method in the modulation plant, described method comprises:
A. introduce recombinant expression cassettes in vegetable cell, described recombinant expression cassettes comprises the polynucleotide claimed in claim 1 that effectively are connected to promotor;
B. under the plant cell growth condition, cultivate described vegetable cell; With
C. from described vegetable cell aftergrowth; Diel rhythm in the wherein said plant is modulated.
12. method according to claim 11, wherein said plant is selected from: corn, soybean, Chinese sorghum, canola oil dish, wheat, clover, cotton, paddy rice, barley, millet, peanut and cocoa.
13. a product that derives from the method that the transgenic plant tissue is processed, the separation polynucleotide of the gene that described transgenic plant tissue expression coding works round the clock, described method comprises:
A. use the recombinant expression cassettes transformed plant cells, described recombinant expression cassettes comprises polynucleotide, described polynucleotide be selected from SEQ ID NO:1,2,3,4,5,6,7,8,20,40,184,186,188,190,192,194,196,198,200,202,204,206,208,210,212,214,216,218,220,222,224,226,228,230,232,234,236,238,240,242,244,246,248,250,252,254,256,258,260,262,264,266,268,270,272,274,276,278,280,282,284,286,288,290,292,294,296,298,300,302,304,306,308,310,312,314,316,318,320,322,324,326,328,330,332,334,336,338,340,342,344,346,348,350,352,354,356,358,360,362,364,366,368,370,372,374,376,378,380,382,384,386,388,390,392,394,396,398,400,402,404,406,408,410,412,414,416,418,420,422,424,426,428,430,432,434,436,438,440,442,444,446,448,450,452,, 454,456,458,460,462,464,466, the full length sequence of 468 and 470 polynucleotide has at least 90% sequence identity; Described polynucleotide are connected to promotor effectively; With
B. under the plant cell growth condition, cultivate the vegetable cell of described conversion; The growth of the vegetable cell of wherein said conversion is modulated;
C. under the condition that forms plant, cultivate described vegetable cell, in plant tissue, to express described polynucleotide; With
D. described plant tissue is processed to obtain product.
14. transgenic plant according to claim 13, wherein said plant is monocotyledons.
15. transgenic plant according to claim 13, wherein said plant is selected from: corn, soybean, Sunflower Receptacle, Chinese sorghum, canola oil dish, wheat, clover, cotton, paddy rice, barley, sugarcane and millet.
16. transgenic plant according to claim 4, cross the expressing of wherein said polynucleotide cause comparing with unconverted plant the plant-growth that has improved.
17. transgenic plant according to claim 4, wherein said plant is compared with unconverted plant, shows the Resource of improvement.
18. transgenic plant according to claim 4, wherein said plant is compared with unconverted plant, has the output of raising.
19. comprising, a regulation and control polynucleotide molecule, described molecule be selected from following sequence: (a) SEQ ID NO:31-183; (b) nucleic acid fragment, described nucleic acid fragment comprise at least 50-100 continuous nucleotide of one of SEQ ID NO:31-183, and wherein said fragment comprises the listed one or more round the clock controlling elements of table 2; (c) nucleotide sequence, described nucleotide sequence have the identity with GAP algorithm that determine and about 500-1000 continuous nucleotide at least 90% one of SEQ ID NO:31-183 under default parameters.
20. a chimeric polynucleotide molecule, described molecule comprise the described nucleic acid fragment of claim 19.
21. chimeric molecule according to claim 20, described molecule comprise described round the clock controlling element and tissue specific expression element.
22. chimeric molecule according to claim 21, wherein said tissue specific expression element is selected from root-specific, vascular bundle sheath cell specificity, leaf specificity and embryo-specific expression element.
23. regulation and control polynucleotide molecule according to claim 19, wherein said regulation and control polynucleotide molecule is promotor.
24. a construct that comprises the described regulatory molecule of claim 19, described regulatory molecule effectively is connected to heterologous polynucleotide molecules, and wherein said heterologous molecule is given the proterties of paying close attention to.
25. construct according to claim 24, the wherein said proterties of paying close attention to is selected from drought tolerance, frost resistance, winter hardiness, disease resistance and insect-resistant.
26. construct according to claim 24, wherein said heterologous molecule works in source-Ku metabolism.
27. transgenic plant that transform with the described regulatory molecule of claim 19.
28. transgenic plant according to claim 27, it is monocotyledons.
29. transgenic plant according to claim 27, it is selected from: corn, soybean, canola oil dish, cotton, Sunflower Receptacle, clover, beet, wheat, naked barley, paddy rice, sugarcane, oat, barley, turfgrass, Chinese sorghum, millet, tomato, pigeonpea, vegetables, fruit tree and forage grass.
30. a method that increases plant biomass, described method are included under the control of the described regulatory molecule of claim 19 and express the heterologous polynucleotide of paying close attention to.
31. method according to claim 30, wherein said heterologous polynucleotide are the plant genes of regulating and control round the clock.
32. a method that improves plant abiotic stress patience, described method are included in and express one or more under the control of the described regulatory molecule of claim 19 give the polynucleotide of abiotic stress patience in plant.
33. method according to claim 32, wherein said abiotic stress patience is selected from drought tolerance, frost resistance and winter hardiness.
34. method according to claim 33, the wherein said polynucleotide of giving drought tolerance are expressed under controlling element control, and its peak value is expressed and betided about noon or dusk.
35. method according to claim 33, the wherein said polynucleotide of giving frost resistance or winter hardiness are expressed under controlling element control, and its peak value is expressed and betided about dawn or morning centre.
36. the method that the output that reduces transgene expression suppresses, described method comprise that expression effectively is connected to the transgenosis of regulation and control polynucleotide molecule, described regulation and control polynucleotide molecule comprises and is selected from following sequence: (a) SEQ ID NO:31-183; (b) nucleic acid fragment, described nucleic acid fragment comprise at least 50-100 continuous nucleotide of one of SEQ ID NO:31-183, and wherein said fragment comprises the listed one or more round the clock controlling elements of table 2; (c) nucleotide sequence, described nucleotide sequence have the identity with GAP algorithm that determine and about 500-1000 continuous nucleotide at least 90% one of SEQ ID NO:31-183 under default parameters.
37. a screening relates to the method for the candidate gene of abiotic stress patience, described method comprises (a) evaluation described candidate gene of expression under the one or more candidate genes that show the output inhibition under constructive expression or the tissue specific expression and the regulatory molecule that (b) is guiding the Circadian Expression pattern are controlled.
38. comprising, described method according to claim 37, wherein said regulatory molecule be selected from following sequence: (a) SEQ ID NO:31-183; (b) nucleic acid fragment, described nucleic acid fragment comprise at least 50-100 continuous nucleotide of one of SEQ ID NO:31-183, and wherein said fragment comprises the listed one or more round the clock controlling elements of table 2; (c) nucleotide sequence, described nucleotide sequence have the identity with GAP algorithm that determine and about 500-1000 continuous nucleotide at least 90% one of SEQ ID NO:31-183 under default parameters.
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